Monomer compositions for the synthesis of RNA, methods of synthesis, and methods of deprotection

ABSTRACT

Nucleotide monomers, polynucleotides, methods of making making each, methods of deprotecting each, and the like are disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to copending U.S. provisional applicationentitled, “METHODS FOR DEPROTECTING POLYNUCLEOTIDES,” having Ser. No.60/731,723, filed Oct. 31, 2005, which is entirely incorporated hereinby reference.

This application is related to copending U.S. Utility patent applicationentitled “MONOMER COMPOSITIONS FOR THE SYNTHESIS OF POLYNUCLEOTIDES,METHODS OF SYNTHESIS, AND METHODS OF DEPROTECTION” filed on Mar. 23,2006 to Dellinger et al. (client matter number 10041386-1) and accordedSer. No. ______, which is entirely incorporated herein by reference.

This application is related to copending U.S. Utility patent applicationentitled “MONOMER COMPOSITIONS FOR THE SYNTHESIS OF POLYNUCLEOTIDES,METHODS OF SYNTHESIS, AND METHODS OF DEPROTECTION” filed on Mar. 23,2006 to Dellinger et al. (client matter number 10051500-2) and accordedSer. No. ______, which is entirely incorporated herein by reference.

This application is related to copending U.S. Utility patent applicationentitled “CLEAVABLE LINKERS FOR POLYNUCLEOTIDES” filed on Mar. 23, 2006to Dellinger et al. (client matter number 10051645-1) and accorded Ser.No. ______, which is entirely incorporated herein by reference.

This application is related to copending U.S. Utility patent applicationentitled “THIOCARBONATE LINKERS FOR POLYNUCLEOTIDES” filed on Mar. 23,2006 to Dellinger et al. (client matter number 10060323-1) and accordedSer. No. ______, which is entirely incorporated herein by reference.

This application is related to copending U.S. Utility patent applicationentitled “PHOSPHORUS PROTECTING GROUPS” filed on Mar. 23, 2006 toDellinger et al. (client matter number 10060321-1) and accorded Ser. No.______, which is entirely incorporated herein by reference.

BACKGROUND

Advances in the chemical synthesis of oligoribonucleotides have not keptpace with the many advances in techniques developed for the chemicalsynthesis of oligodeoxyribo-nucleotides. The synthesis of RNA isactually a much more difficult task than the synthesis of DNA. Theinternucleotide bond in native RNA is far less stable than in the DNAseries. The close proximity of a protected 2′-hydroxyl to theinternucleotide phosphate presents problems, both in terms of theformation of the internucleotide linkage and in the removal of the2′-protecting group once the oligoribonucleotide has been synthesized(See FIG. 1).

Only recently has there been a great demand for small synthetic RNA. Thediscoveries of the RNAi pathway and small RNAs, such as siRNA, miRNAsand ntcRNAs associated with the RNA interference pathway is primarilyresponsible for this increased demand. Most recent attempts at thechemical synthesis of oligoribonucleotides have followed the syntheticstrategy for the chemical synthesis of oligodeoxyribonucleotides: thestandard phosphoramidite approach [Matteucci, M. D., Caruthers, M. H. JAm. Chem. Soc. 1981, 103, 3186-3191]. Such methods proceed by thestep-wise addition of protected ribonucleoside phosphoramidite monomersto a growing RNA chain connected to a solid phase support. However,efficient solid phase synthesis of oligoribonucleotides still poorlycompared to the efficiency of oligodeoxyribonucleotides synthesis.

Until recently, the typical approach to RNA synthesis utilized monomerswhereby the 5′-hydroxyl of the ribonucleoside was protected by theacid-labile dimethoxytrityl (DMT) protecting group. Various protectinggroups have been placed on the 2′-hydroxyl to prevent isomerization andcleavage of the intemucleotide bond during the acid deprotection step.By using this as a starting point for RNA synthesis, researchers havefocused on finding an ideal 2′-protecting group compatible with aciddeprotection. Research directed toward the discovery of this ideal2′-protecting group has taken two primary courses: the use ofacid-stable 2′-protecting groups and the use of acid-labile2′-protecting groups. The use of acid-stable 2′-protecting groups hasbeen quite limiting from a chemical perspective, since there are notmany options available when the base lability of RNA is considered.Acid-stable protecting groups are typically base-labile ornucleophile-labile (e.g., removed by a strong base or a strongnucleophile). General base-labile protecting groups are removed byelimination or fragmentation subsequent to proton abstraction by astrong base. An example of this type of protecting group is apropionitrile-containing protecting group, which is removed bybeta-elimination to form acrylonitrile after a proton is abstracted fromthe methylene carbon adjacent to the nitrile group. It is difficult touse these types of protecting groups on the 2′-hydroxyl of RNA sincesubsequent proton abstraction from the ensuing 2′-hydroxyl results incleavage of the intemucleotide bond via formation of a 2′-3′ cyclicphosphate and destruction of the RNA.

This approach is therefore only viable if the pH conditions used forproton abstraction from the protecting group are below pH 11, the pH atwhich proton abstraction from the 2′-hydroxyl begins to give rapidcleavage of the intemucleotide bond. The approach of using a generalbase-labile protecting group for the 2′-hydroxyl has been furtherstymied by the necessary use of weak bases during the oxidation andcapping reactions that occur in the standard phosphoramiditeoligonucleotide synthesis process.

Protecting groups that are removed by the weakly basic conditions belowpH 11 (such that the 2′-hydroxyl is not appreciably deprotonated) aretypically unstable to the conditions used for capping and oxidation. Asa result, the approach of using general base-labile protecting groupsfor 2′-hydroxyl protection has rarely been pursued, and never enabled.

Alternatively, there have been many attempts at the use ofnucleophile-labile protecting groups for the protection of the2′-hydroxyl. The difficulty associated with the use ofnucleophile-labile protecting groups is that most typical nucleophilesare governed by the Brønsted-type plot of nucleophilicity as a functionof basicity: the stronger the nucleophilicity, the stronger thebasicity. As a result, strong nucleophiles are usually also strong basesand therefore the use of strong nucleophiles for deprotection of the2′-hydroxyl typically results in the destruction of the desired RNAproduct by a subsequent proton abstraction from the 2′-hydroxyl. The useof nucleophile-labile 2′-hydroxyl protecting groups for RNA synthesishas only been enabled by the use of fluoride ion, a silicon-specificnucleophile that is reactive with silanes and siloxanes at a widevariety of pH conditions.

The most popular of these acid-stable protecting groups seem to be thet-butyl-dimethylsilyl group known as TBDMS [Ogilvie et al., Can. J.Chem., Vol 57, pp. 2230-2238 (1979)]. Widely practiced in the researchcommunity, the use of TBDMS as 2′-protecting group, dominated thepreviously small market for chemical synthesis of RNA for a very longtime [Usman et al. J. Am. Chem. Soc. 109 (1987) 7845], [Ogilvie et al.Proc. Natl. Acad. Sci. USA 85 (1988) 5764]. The oligoribonucleotidesyntheses carried out therewith are, however, by no means satisfactoryand typically produces poor quality RNA products.

Several publications have reported the migration of the alkylsilyl groupunder a variety of conditions [Scaringe et al, Nucleic Acids Res 18,(18) 1990 5433-5441; Hogrefe et al. Nucleic Acids Research, 1993, 21(20), 4739-4741]. Also, the loss of the 2′-silyl group that occursduring the removal of exocyclic amine protecting groups has been widelydescribed in the literature [Stawinski et. al. Nucleic Acids Res. 1988,16 (19), 9285-9298]. Methods that use less stable exocyclic amineprotecting groups such as phenoxyacetyl or methoxyacetyl weresubsequently developed to circumvent this problem [Schulhof et al.Nucleic Acids Res. 1987 15(2) 397-416]. However, the synthesis of the5′-O-dimethoxytrityl-2′-O-tert-butyldimethylsilyl-ribo-3′-O-(β-cyanoethyl,N-diisopropyl)phosphoramidite monomers is still challenging and costlydue to the non-regioselective introduction of the 2′-silyl group and theadded chemical requirements to prevent migration of the silyl groupduring the phosphoramidite production. It is also well known in the artthat the coupling efficiency of these monomers is greatly decreased dueto the steric hindrance of the 2′-TBDMS protecting group, therebyaffecting the yield and purity of the full-length product, and alsolimiting the length of the oligoribonucleotide that can be achieved bythis chemical synthesis.

The most recent acid-stable 2′-hydroxyl protection approach for RNAsynthesis was developed by Pitsch et al. [U.S. Pat. No. 5,986,084] totry to circumvent the problems encountered with the previous 2′-silylprotecting groups. This approach also relies on the use of 2′-O-acetalsgroups further protected by an alkylsilane, which is removed by thesilicon-specific nucleophile fluoride ion. Although somewhat less acidstable than TBDMS, it is used in combination with acid-labile5′-protecting groups such as DMT or the 5′-9-phenylxanthen-9-yl (Pix)group shown below.

Because of the presence of the methylene group, this 2′-protecting groupis less bulky than the TBDMS, allowing higher coupling efficiency. Sincethe protecting group is an acetal moiety, there is no significantproblem of isomerization. The commercial protecting group typically usedin this approach is the tri-isopropyloxymethyl derivative known by theabbreviation TOM. Although this protecting group scheme solves many ofthe problems encountered by the TBDMS chemistry, it suffers from othersignificant difficulties. The synthesis of the TOM-protected monomers isextremely difficult and low yielding. The protecting group itselfrequires a low yield multi-step synthesis prior to its placement on thenucleoside. The attachment to the nucleoside is performed through anucleophillic displacement reaction by a 2′-3′ alkoxide generated from adialkyl tin reagent that produces a mixture of non-regioselectiveproducts that have to be separated and isolated by chromatography. Inthe case of the guanosine nucleoside, the tin reagents canpreferentially react with the heterobase rather than the 2′-,3′-hydroxyl moieties. In many cases, the overall yield of desiredproducts from these reactions can be significantly less than 10%,rendering the monomer synthons and subsequent RNA products veryexpensive to produce.

Alternatively, many researchers have pursued the use of acid-labilegroups for the protection of the 2′-hydroxyl moiety. The classicacid-labile protecting group is the 2′-acetal moiety, which wasinitially developed by Reese [Reese, C. B., Org. Biomol. Chem. 2005,3(21), 3851-68], such as tetrahydropyran (THP) or4-methoxy-tetrahydropyran (MTHP), 1-(2-chloroethoxy)ethyl (Cee) [O.Sakatsume et al. Tetrahedron 47 (1991) 8717-8728],1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp) [M. Vaman Rao et al.,J. Chem. Soc. Perkin Trans., Paper 2:43-55 (1993), Daniel C. Capaldi etal., Nucleic Acids Research, 22(12):2209-2216 (1994)].

One of the advantages of acetal protecting groups compared with silylethers protecting groups is that they can be introduced regioselectivelyinto the 2′ position through the use of the Markiewicz protecting group:tetraisopropyldisiloxane-1,3-diyl. This protecting group, also known asTIPS, simultaneously blocks the 5′- and 3′-hydroxyls to allow completeregioselective upon introduction of the acetal group on to the2′-hydroxyl [Markiewicz W. T., J. Chem Research (S) 1979 24-25)].Another advantage is that the phosphoramidite coupling with 2′-acetalprotected monomers is typically more efficient than with trialkylsilanes. The problems encountered when using the combination of 5′-O-DMTand 2′-O-acetals groups reside in the difficulty to find suitable 2′-Oacetal groups that are both completely stable to the anhydrous acidicconditions used to remove the 5′O-DMT group and completely labile to themild aqueous acid conditions used to remove this 2′-acetal protectinggroup, while not cleaving the internucleotide bond of the RNA. Theremoval of acetals that are stable under DMT deprotection conditionstypically requires prolonged exposure to acidic conditions that degradethe RNA. To inhibit the loss of the 2′protecting group, the5′-9-phenylxanthen-9-yl (Pix) group was applied, which is more labilethan the DMT protecting group.

Even considering all of these innovations, the inability to find aviable combination of 2′-acetal and 5′-acid labile protecting groupsthat fits into the standard phosphoramidite synthesis cycle has resultedin these chemical schemes that were never effectively commercialized.Conversely, acetals used in combination with 5′ protecting groups suchas leuvinyl and 9-fluorenylmethyloxycarbonyl (FMOC) that are deprotectedunder non-acidic conditions like hydrazinolysis have not met significantsuccess. One of the overriding reasons that 2′-acetals have not achievedwide acceptance is that they tend to be too stable under the requiredacid deprotection conditions once the monomers are incorporated onto anoligonucleotide, due to the close proximity of the protected 2′-hydroxylto the internucleotide phosphate. There is a significant change in thestability of the protecting group once the oligonucleotide is produced.Conditions that can effectively remove an acetal group from a protectednucleoside monomer tend to be ineffective to remove the same group fromthe oligonucleotide.

To address this issue, Dellinger et al. developed 2′-orthoesterprotecting groups whose labiality on the oligonucleotide is lessaffected by close proximity to the internucleotide phosphate allowingeffective removal under aqueous acid conditions that do not degrade thedesired RNA product. The use of 2′-cyclic orthoesters was evaluatedusing a regioselective coupling procedure as well as a set of5′-nucleophile labile carbonates [Marvin H. Caruthers, Tadeusz K.Wyrzkiewicz, and Douglas J. Dellinger. “Synthesis of Oligonucleotidesand Oligonucleotide Analogs on Polymer Supports” In Innovation andPerspectives in Solid Phase Synthesis: Peptides, Proteins and NucleicAcids (R. Epton, ed.) Mayflower Worldwide Limited, Birmingham, 39-44(1994)]. Subsequently, Scaringe et. al. developed a set of 5′- and2′-protecting groups that overcome the problems associated with use of5′-DMT. This method uses a 5′-silyloxy protecting group [patents U.S.Pat. No. 5,889,136, U.S. Pat. No. 6,111,086, and U.S. Pat. No.6,590,093] which require silicon-specific fluoride ion nucleophiles tobe removed, in conjugation with the use of optimized 2′-orthoestersprotecting groups (ACE). Although the coupling efficiency is greatlyincreased with the use of the ACE 2′-orthoester protecting group, andthe final deprotection facile under pH conditions at which RNA isstable, the use of fluoride anions to deprotect the 5′-protecting groupsprior each condensation cycle carries some disadvantages for routinesynthesis of RNA and is even more problematic for large-scale synthesisof RNA. Because this chemistry requires atypical nucleoside protectinggroups and custom synthesized monomers, namely on the 5′OH, it isdifficult and time consuming to build RNA sequences that contain othercommercially available phosphoramidite monomers, such as modifiednucleotides, fluorescent labels, or anchors.

In order to incorporate a wide variety of alternative monomers andmodifications using this chemistry, it is necessary to have each of themcustom-synthesized with the appropriate 5′-silyloxy protecting group,thus significantly limiting the commercial applications for thischemistry. The ACE chemistry has the ability to produce very highquality RNA, but the reactions conditions are tricky and the synthesisnot robust enough to routinely produce long sequences of RNAs. As aresult, there is still clearly a need for the development of a chemicalsynthesis method for RNA that is simple and robust and produces highquality RNA products, while fitting into the standard phosphoramiditeoligonucleotide synthesis approach. The commercial success of the ACEchemistry clearly illustrates the need to develop a RNA synthesis methodthat is founded upon mild and simple final deprotection conditions thatwill not affect the integrity of the final RNA product.

While protected, the RNA molecule has similar stability to the DNAmolecule. Consequently, the final deprotection conditions to treat asynthetic RNA molecule are typically the same as the conditions to treata synthetic DNA molecule prior to the removal of the 2′-hydroxylprotecting group. As a result, the current methods of RNA synthesisperform the final deprotection of the synthetic RNA in a 4-, 3-, or a2-step fashion.

-   -   1. Deprotection of the protected phosphotriester, most commonly        the cyanoethyl group (CNE), which is performed by brief exposure        to ammonia (½ hr at room temperature) or in the case of the        methyl group, by treatment with thiophenol for ½ hr at room        temperature.    -   2. Cleavage of the oligoribonucleotides from the support        performed under basic conditions, usually by exposure to        ammonium hydroxide, anhydrous ammonia in an alcohol,        methylamine, other alkyl amines, basic non-amine solutions such        as potassium carbonate solutions, or non-nucleophillic bases        such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in organic        solvents.    -   3. Deprotection of the nucleobases, which one most commonly        protected on the exocyclic amine with protecting groups such as        phenoxyacetyl (PAC), acetyl (Ac), isobutyryl (iBu) or benzoyl        (Bz) and which are typically also removed under basic        conditions. Most of the time, steps 2 and 3 are performed        simultaneously.    -   4. Usually, the 2′-deprotection is performed post-cleavage of        the oligonucleotide from the support. In the case of TBDMS and        TOM chemistry, the oligoribonucleotide is exposed to fluoride        anions to deblock the 2′ hydroxyl groups after cleavage of the        oligoribonucleotide from the support. In the case of the ACE        chemistry, the 2′-O-orthoester group can be removed with acidic        conditions after cleavage of the oligoribonucleotide from the        support, but also can be kept and deprotected during shipment to        the customers or after shipment by the customers (this procedure        allows keeping the oligoribonucleotide intact longer, since RNA        is very sensitive to nuclease RNase degradation).        Steps 1-3 can be performed simultaneously, when appropriate,        making it a 2-step deprotection process, or step 1 can be        performed independently, and steps 2-3 combined, making it a        3-step final deprotection process.

The removal of the 2′-hydroxyl protecting group is problematic for boththe 3- and 2-step processes. In the 3-step process, the phosphorusprotecting group is typically removed first, while theoligoribonucleotide is still attached to a solid support. In the secondstep, the heterobase protecting groups are removed using a nucleophillicbase like ammonia or methyl amine, also which usually result in thecleavage of the oligoribonucleotide from the support.

Finally, a fluoride ion-based solution under neutral, mildly acidic, ormildly basic conditions (TBDMS, TOM) [Pitsch, et. al. Helv. Chim. Acta,2001, 84, 3773-3795] or a weak acidic solution is used to remove the ACE2′-hydroxyl protecting group [Scaringe et al, Nucleic Acids Res 18, (18)5433-5441 (1990); Scaringe et al, J. Am. Chem. Soc., 120, 11820-11821(1998)]. This process requires that the 2′-hydroxyl protecting group isorthogonally stable to the deblock conditions utilized to remove theprotecting group for the 3′- or 5′-hydroxyl functional during thechemical synthesis process, and stable to the conditions utilized fordeprotection of the phosphorus protecting groups and the heterobaseprotecting groups. Most often it is seen that a loss of the2′-protecting group occurs to some extent during one of these previousdeblock or deprotection processes. The result is modification of thedesired RNA strand or cleavage of the desired RNA product.

Modification and cleavage decreases the yield and quality of the desiredRNA products and can often prevent synthesis and isolation ofoligonucleotide sequences significantly longer than 15 or 20 nucleotidesin length. In the case of the use of a fluoride ion solution fordeprotection of the 2′-hydroxy group, removal of residual fluoride ionsrequires additional steps and can be quite difficult and time consuming.

In the 3-step process, removal of the phosphorus protecting groups isaccomplished simultaneously with the removal of the heterobaseprotecting groups. This is usually accomplished using a nucleophillicbase like ammonia or methylamine. Most often the phosphorus-protectinggroup is removed using a beta-elimination reaction such as the formationof acrylonitrile from a 3-hydroxypropionitrile ester. However, the useof this system for the protection of the internucleotide phosphodiesterlinkage, followed by simultaneous deprotection during cleavage of theheterobase protecting groups, results in a number of notable sidereactions that affect the yield and purity of the final product. The useof protecting groups that are susceptible to cleavage by protonabstraction followed by beta-elimination generally decreases thereactivity of the active phosphorus intermediate due to their electronwithdrawing nature, and this effect lowers the per-cycle couplingefficiency. In addition, the elimination products such as acrylonitrileare reactive toward the heterobases and often form base adducts thatresult in undesired modifications.

SUMMARY

Nucleotide monomers, polynucleotides, methods of making making each,methods of deprotecting each, and the like are disclosed herein. Anembodiment of a nucleotide monomer, among others, includes a structure Ias described herein.

Another embodiment of a nucleotide monomer, among others, includes astructure II as described herein.

Another embodiment of the polynucleotide, among others, includes apolynucleotide that include combinations of nucleotide moieties such as,but not limited to, structure I, structure II, nucleotide moities havingone or more protecting groups as described herein, nucleotide moietiesnot including protecting groups, nucleotide moieties knowon the the art,combinations thereof, and the like.

An embodiment of deprotecting polynucleotides, among others, includes:providing a polynucletide including combinations of nucleotide moietiessuch as, but not limited to, structure I, structure II, nucleotidemoities having one or more protecting groups as described herein,nucleotide moieties not including protecting groups, nucleotide moietiesknowon the the art, combinations thereof, and the like; and deprotectingthe 2′-hydroxyl protecting group and the exocyclic amino protectinggroup in a solution including an α-effect nucleophile, wherein thesolution is at a pH of about 4 to 10, wherein the α-effect nucleophilehas a pKa of about 4 to 13.

An embodiment of deprotecting polynucleotides, among others, includes:synthesizing on a solid support a polynucletide including combinationsof nucleotide moieties such as, but not limited to, structure I,structure II, nucleotide moities having one or more protecting groups asdescribed herein, nucleotide moieties not including protecting groups,nucleotide moieties knowon the the art, combinations thereof, and thelike; introducing the polynucleotide to a solution including an α-effectnucleophile, wherein the solution is at a pH of about 6 to 11, whereinthe α-effect nucleophile has a pKa of about 4 to 13; and deprotectingthe 2′-hydroxyl protecting group.

Additional objects, advantages, and novel features of this disclosureshall be set forth in part in the descriptions and examples that followand in part will become apparent to those skilled in the art uponexamination of the following specifications or may be learned by thepractice of the disclosure. The objects and advantages of the disclosuremay be realized and attained by means of the instruments, combinations,compositions, and methods particularly pointed out in the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference is now made to the following drawings. Note that thecomponents in the drawings are not necessarily to scale.

FIG. 1 schematically illustrates a prior art multi-step RNA synthesismethod.

FIGS. 2A through 2E illustrate chromatograph of a synthetic RNA and asolution of 5% hydrogen peroxide in a solution having a pH of about 9 atvarious times (FIG. 2A (time_(RNA)=0), FIG. 2B (time_(HP)=0), FIG. 2C(time=3 hours), FIG. 2D (time=12 hours), and FIG. 2E (time=24 hours)).

FIG. 3 illustrates the transient protection of hydroxyl moieties (“JonesProcedure”).

FIG. 4 illustrates the transient protection of hydroxyl moieties(“Markiewicz Procedure”).

FIG. 5 illustrates the selective protection of exocyclic amine withchloroformate reagents.

FIG. 6 illustrates the selective protection of 2′-hydroxyl withchloroformate reagents.

FIG. 7 illustrates the preparation of5′,3′-O-(tetraisopropyldisiloxane-1,3-diyl) ribonucleosides.

FIG. 8 illustrates the simultaneous protection of5′,3′-O-(tetraisopropyldisiloxane-1,3-diyl) ribonucleosides usingchloroformates or pyrocarbonates.

FIG. 9 illustrates the selective protection of exocyclic amine withpyrocarbonate reagents.

FIG. 10 illustrates the selective protection of 2′-hydroxyl withpyrocarbonate reagents.

FIG. 11 illustrates the selective protection of exocyclic amine withhemimethylthioacetal chloroformate reagents.

FIG. 12 illustrates the synthesis ofO-trimethylsilylhemimethylthioacetal as an intermediate in thepreparation of the corresponding chloroformate.

FIG. 13 illustrates the O-trimethylsilylhemimethylthioketals asintermediate in the preparation of the corresponding pyrocarbonate.

FIG. 14 illustrates the selective protection of exocyclic amine withhemimethylthioketal pyrocarbonate reagents.

FIG. 15 illustrates the selective protection of 2′-hydroxyl withhemimethylthioketal pyrocarbonate reagents.

FIG. 16 illustrates the 2′-hydroxyl protective groups.

FIG. 17 illustrates the Michael addition at the C-6 carbon of theheterobase followed by nucleophillic acyl substitution at the C-4 carbonresulting in formation of a urea.

FIG. 18 illustrates that O-4 protection prevents initial Michaeladdition at C-6.

FIG. 19 illustrates the formation of C-4 triazolide.

FIG. 20 illustrates the regiospecific synthesis of a 2′-protectednucleoside with O-4 protection.

FIG. 21 illustrates HPLC Chromatograms of RNA synthesized by the presentdisclosure.

FIG. 22 illustrates HPLC Chromatograms of RNA synthesized by the presentdisclosure.

FIG. 23 illustrates HPLC Chromatograms of RNA synthesized by the presentdisclosure.

DETAILED DESCRIPTION

Embodiments of the present disclosure employ, unless otherwiseindicated, conventional techniques of synthetic organic chemistry,biochemistry, molecular biology, and the like, which are within theskill of one in the art. Such techniques are explained fully in theliterature.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions disclosed and claimedherein. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for in the specification. Unlessindicated otherwise, parts are parts by weight, temperature is in ° C.,and pressure is at or near atmospheric. Standard temperature andpressure are defined as 20° C. and 1 atmosphere.

Before the embodiments of the present disclosure are described indetail, it is to be understood that unless otherwise indicated, thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such mayvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps may be executed in different sequence where this is logicallypossible.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meanings,unless a contrary intention is apparent.

As used herein, polynucleotides include single or multiple strandedconfigurations, where one or more of the strands may or may not becompletely aligned with another. The terms “polynucleotide” and“oligonucleotide” shall be generic to polydeoxynucleotides (containing2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to anyother type of polynucleotide which is an N-glycoside of a purine orpyrimidine base, and to other polymers in which the conventionalbackbone has been replaced with a non-naturally occurring or syntheticbackbone or in which one or more of the conventional bases has beenreplaced with a non-naturally occurring or synthetic base.

A “nucleotide” and a “nucleotide moiety” refer to a sub-unit of anucleic acid (whether DNA or RNA or an analogue thereof) which mayinclude, but is not limited to, a phosphate group, a sugar group and anitrogen containing base, as well as analogs of such sub-units. Othergroups (e.g., protecting groups) can be attached to the sugar group andnitrogen containing base group.

A “nucleoside” references a nucleic acid subunit including a sugar groupand a nitrogen containing base. It should be noted that the term“nucleotide” is used herein to describe embodiments of the disclosure,but that one skilled in the art would understand that the term“nucleoside” and “nucleotide” are interchangable in most instances. Oneskilled in the art would have the understanding that additionalmodification to the nucleoside may be necessary, and one skilled in theart has such knowledge.

A “nucleotide monomer” refers to a molecule which is not incorporated ina larger oligo- or poly-nucleotide chain and which corresponds to asingle nucleotide sub-unit; nucleotide monomers may also have activatingor protecting groups, if such groups are necessary for the intended useof the nucleotide monomer.

A “polynucleotide intermediate” references a molecule occurring betweensteps in chemical synthesis of a polynucleotide, where thepolynucleotide intermediate is subjected to further reactions to get theintended final product (e.g., a phosphite intermediate, which isoxidized to a phosphate in a later step in the synthesis), or aprotected polynucleotide, which is then deprotected.

An “oligonucleotide” generally refers to a nucleotide multimer of about2 to 100 nucleotides in length, while a “polynucleotide” includes anucleotide multimer having any number of nucleotides greater than 1. Theterms “oligonucleotide” and “polynucleotide” are often usedinterchangeably, consistent with the context of the sentence andparagraph in which they are used in.

It will be appreciated that, as used herein, the terms “nucleoside” and“nucleotide” will include those moieties which contain not only thenaturally occurring purine and pyrimidine bases, e.g., adenine (A),thymine (T), cytosine (C), guanine (G), or uracil (U), but also modifiedpurine and pyrimidine bases and other heterocyclic bases which have beenmodified (these moieties are sometimes referred to herein, collectively,as “purine and pyrimidine bases and analogs thereof”). Suchmodifications include, e.g., diaminopurine and its deravitives, inosineand its deravitives, alkylated purines or pyrimidines, acylated purinesor pyrimidines thiolated purines or pyrimidines, and the like, or theaddition of a protecting group such as acetyl, difluoroacetyl,trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl,phenoxyacetyl, dimethylformamidine, N,N-diphenyl carbamate, or the like.The purine or pyrimidine base may also be an analog of the foregoing;suitable analogs will be known to those skilled in the art and aredescribed in the pertinent texts and literature. Common analogs include,but are not limited to, 1-methyladenine, 2-methyladenine,N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine,N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine,5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine,2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil,5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil,5-(carboxymethylaminomethyl)-uracil, 2-thiouracil,5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, pseudouracil,1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine,xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine, and2,6-diaminopurine.

An “intemucleotide bond” refers to a chemical linkage between twonucleoside moieties, such as a phosphodiester linkage in nucleic acidsfound in nature, or such as linkages well known from the art ofsynthesis of nucleic acids and nucleic acid analogues. An intemucleotidebond may include a phospho or phosphite group, and may include linkageswhere one or more oxygen atoms of the phospho or phosphite group areeither modified with a substituent or replaced with another atom, e.g.,a sulfur atom, or the nitrogen atom of a mono- or di-alkyl amino group.

A “group” includes both substituted and unsubstituted forms. Typicalsubstituents include one or more lower alkyl, amino, imino, amido,alkylamino, arylamino, alkoxy, aryloxy, thio, alkylthio, arylthio,oraryl, or alkyl; aryl, alkoxy, thioalkyl, hydroxyl, amino, amido,sulfonyl, thio, mercapto, imino, halo, cyano, nitro, nitroso, azido,carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, andboronyl, or optionally substituted on one or more available carbon atomswith a nonhydrocarbyl substituent such as cyano, nitro, halogen,hydroxyl, sulfonic acid, sulfate, phosphonic acid, phosphate,phosphonate, or the like. Any substituents are typically chosen so asnot to substantially adversely affect reaction yield (for example, notlower it by more than 20% (or 10%, or 5%, or 1%) of the yield otherwiseobtained without a particular substituent or substituent combination).An “acetic acid” includes substituted acetic acids such asdi-chloroacetic acid (DCA) or tri-chloroacetic acid (TCA).

A “phospho” group includes a phosphodiester, phosphotriester, andH-phosphonate groups. In the case of either a phospho or phosphitegroup, a chemical moiety other than a substituted 5-membered furyl ringmay be attached to O of the phospho or phosphite group which linksbetween the furyl ring and the P atom.

A “protecting group” is used in the conventional chemical sense toreference a group, which reversibly renders unreactive a functionalgroup under specified conditions of a desired reaction, as taught, forexample, in Greene, et al., “Protective Groups in Organic Synthesis,”John Wiley and Sons, Second Edition, 1991, which is incorporated hereinby reference. After the desired reaction, protecting groups may beremoved to deprotect the protected functional group. All protectinggroups should be removable (and hence, labile) under conditions which donot degrade a substantial proportion of the molecules being synthesized.In contrast to a protecting group, a “capping group” permanently bindsto a segment of a molecule to prevent any further chemicaltransformation of that segment. It should be noted that thefunctionality protected by the protecting group may or may not be a partof what is referred to as the protecting group.

A “hydroxyl protecting group” or “O-protecting group” refers to aprotecting group where the protected group is a hydroxyl. A“reactive-site hydroxyl” is the terminal 5′-hydroxyl during 3′-5′polynucleotide synthesis and is the 3′-hydroxyl during 5′-3′polynucleotide synthesis. An “acid-labile protected hydroxyl” is ahydroxyl group protected by a protecting group that can be removed byacidic conditions. Similarly, an “acid-labile protecting group” is aprotecting group that can be removed by acidic conditions.

A “linking moiety” is a group known in the art to connect nucleotidemoieties in a polynucleotide or oligonucleotide compound.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In certain embodiments,a straight chain or branched chain alkyl has about 30 or fewer carbonatoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ forbranched chain), and alternatively, about 20 or fewer. For example theterm “alkyl” can refer to straight or branched chain hydrocarbon groups,such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl,pentyl, hexyl, heptyl, octyl, and the like. Likewise, cycloalkyls havefrom about 3 to about 10 carbon atoms in their ring structure, andalternatively about 5, 6 or 7 carbons in the ring structure. The term“alkyl” is also defined to include halosubstituted alkyls and heteroatomsubstituted alkyls.

Moreover, the term “alkyl” (or “lower alkyl”) includes “substitutedalkyls”, which refers to alkyl moieties having substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone. Suchsubstituents may include, for example, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphonate, a phosphinate, an amino, an amido, anamidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, analkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclic, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain may themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CN, and thelike. Cycloalkyls may be further substituted with alkyls, alkenyls,alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, —CN, andthe like.

The term “alkoxy” means an alkyl group linked to oxygen thus: R—O—. Inthis function, R represents the alkyl group. An example would be themethoxy group CH₃O—.

The term “aryl” refers to 5-, 6-, and 7-membered single-ring aromaticgroups that may include from zero to four heteroatoms, for example,benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, andthe like. Those aryl groups having heteroatoms in the ring structure mayalso be referred to as “aryl heterocycles” or “heteroaromatics.”

The aromatic ring may be substituted at one or more ring positions withsuch substituents as described above, for example, halogen, azide,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino,nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone,aldehyde, ester, heterocyclic, aromatic or heteroaromatic moieties,—CF₃, —CN, or the like.

The term “aryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (the rings are “fused rings”) wherein at least one of the rings isaromatic (e.g., the other cyclic rings may be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, and/or heterocyclyls).

The terms “halogen” and “halo” refer to fluorine, chlorine, bromine, andiodine.

The terms “heterocycle”, “heterocyclic”, “heterocyclic group” or“heterocyclo” refer to fully saturated or partially or completelyunsaturated, including aromatic (“heteroaryl”) or nonaromatic cyclicgroups (for example, 3 to 13 member monocyclic, 7 to 17 member bicyclic,or 10 to 20 member tricyclic ring systems) which have at least oneheteroatom in at least one carbon atom-containing ring. Each ring of theheterocyclic group containing a heteroatom may have 1, 2, 3, or 4heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfuratoms, where the nitrogen and sulfur heteroatoms may optionally beoxidized and the nitrogen heteroatoms may optionally be quaternized. Theheterocyclic group may be attached at any heteroatom or carbon atom ofthe ring or ring system. The rings of multi-ring heterocycles may beeither fused, bridged and/or joined through one or more spiro unions.

The terms “substituted heterocycle”, “substituted heterocyclic”,“substituted heterocyclic group” and “substituted heterocyclo” refer toheterocycle, heterocyclic, and heterocyclo groups substituted with oneor more groups preferably selected from alkyl, substituted alkyl,alkenyl, oxo, aryl, substituted aryl, heterocyclo, substitutedheterocyclo, carbocyclo (optionally substituted), halo, hydroxy, alkoxy(optionally substituted), aryloxy (optionally substituted), alkanoyl(optionally substituted), aroyl (optionally substituted), alkylester(optionally substituted), arylester (optionally substituted), cyano,nitro, amido, amino, substituted amino, lactam, urea, urethane,sulfonyl, and the like, where optionally one or more pair ofsubstituents together with the atoms to which they are bonded form a 3to 7 member ring.

When used herein, the terms “hemiacetal”, “thiohemiacetal”, “acetal”,and “thioacetal”, are recognized in the art, and refer to a chemicalmoiety in which a single carbon atom is geminally disubstituted witheither two oxygen atoms or a combination of an oxygen atom and a sulfuratom. In addition, when using the terms, it is understood that thecarbon atom may actually be geminally disubstituted by two carbon atoms,forming ketal, rather than acetal, compounds.

The term “electron-withdrawing group” is art-recognized, and refers tothe tendency of a substituent to attract valence electrons fromneighboring atoms (i.e., the substituent is electronegative with respectto neighboring atoms). A quantification of the level ofelectron-withdrawing capability is given by the Hammett sigma constant.This well known constant is described in many references, for instance,March, Advanced Organic Chemistry 251-59, McGraw Hill Book Company, NewYork, (1977). Exemplary electron-withdrawing groups include nitro, acyl,formyl, sulfonyl, trifluoromethyl, cyano, chloride, and the like.

The term “electron-donating group” is art-recognized, and refers to thetendency of a substituent to repel valence electrons from neighboringatoms (i.e., the substituent is less electronegative with respect toneighboring atoms). Exemplary electron-donating groups include amino,methoxy, alkyl (including C₁₋₆ alkyl that can have a linear or branchedstructure), C₄₋₉ cycloalkyl, and the like.

The term “deprotecting simultaneously” refers to a process which aims atremoving different protecting groups in the same process and performedsubstantially concurrently or concurrently. However, as used herein,this term does not imply that the deprotection of the differentprotecting groups occur at the same time or with the same rate or samekinetics.

As used herein, “dissociation constant” (e.g., an acid dissociationconstant) has its conventional definition as used in the chemical artsand references a characteristic property of a molecule having a tendencyto lose a hydrogen ion. The value of a dissociation constant mentionedherein is typically expressed as a negative log value (i.e., a pKd).

Discussion

The present disclosure includes nucleotide monomers and polynucleotidestructures (e.g., synthetic ribonucleic acid (RNA)) including nucleotidemoieties, where the nucleotide monomers and nucleotide moieties eachinclude various types of protecting groups. The nucleotide monomers andnucleotide moieties can be used in conjunction with methods, processes,and/or compositions of the present disclosure, for the deprotection ofpolynucleotides. Embodiments of the present disclosure enablequantitative or quasi-quantitative and rapid synthesis of the desireddeprotected full-length polynucleotide product.

Embodiments of the present disclosure also include methods, processes,compositions, and structures that enable the synthesis ofpolynucleotides (e.g., RNA) with greater efficiency and lower costcompared to previous methods. In particular, the present disclosureprovides for methods, processes, compositions, and structures thatovercome at least some of the degradation problems of polynucleotides(e.g., RNA) that occurs during the deprotection procedure due to the useof strongly basic conditions, by the use of peroxyanions in mildly basicsolutions and protecting groups described herein (e.g., 2′-hydroxylprotecting groups, base protecting groups, and phosphorus protectinggroups).

An advantage of an embodiment of the present disclosure is that thedeprotection of the bases can be performed even after removal of the2′-hydroxyl protecting groups as opposed to prior methods. Anotheradvantage of an embodiment of the present disclosure is that thedeprotection can be used also to cleave the protecting groups of thebases in polynucleotide synthesis where for example theoligodeoxynucleotide includes a nucleotide or a modified nucleoside, ora non-nucleoside moiety such as fluorescent labels that are sensitive tostrong bases.

An exemplary method of deprotecting polynucleotides, among others,includes: providing a synthetically made ribonucleic acid (RNA) (e.g.,synthesized on a solid support (e.g., bead, CPG, polymeric support, anarray)), wherein the RNA, optionally, has at least one 2′-hydroxylprotecting group (e.g., a silyl protecting group, a silyloxy protectinggroup, a ester protective group, a carbonate protecting group, athiocarbonate protecting group, a carbamate protecting group, an acetalprotecting group, an acetaloxycarbonyl protecting group, an orthoesterprotecting group, an orthothioester protecting group, anorthoesteroxycarbonyl protecting group, orthothioesteroxycarbonylprotecting group, orthoesterthiocarbonyl protecting group,orthothioesterthiocarbonyl protecting group, a thioacetal protectinggroup, a thioacetaloxycarbonyl protecting group, as well as other groupsdescribed herein, and combinations thereof), wherein the RNA,optionally, has at least one protected exocyclic amine on a heterocyclicbase protected by a base protecting group (e.g., an acyl protectivegroup, an oxycarbonyl protecting group, a thiocarbonyl protecting group,an alkyloxymethylcarbonyl protecting group (optionally substituted), analkylthiomethylcarbonyl protecting group (optionally substituted), anaryloxymethylcarbonyl protecting group (optionally substituted), anarylthiomethylcarbonyl protecting group (optionally substituted), andialkylformamidine protecting group (optionally substituted), adialkylamidine protecting group (optionally substituted), as well asother groups described herein, and combinations thereof), wherein theRNA, optionally, has at least one protected imine on a heterocyclic baseby a base protecting group (e.g., an acyl protecting group (optionallysubstituted), an alkyloxylcarbonyl protecting group (optionallysubstituted), an aryloxycarbonyl protecting group (optionallysubstituted), an alkylthiocarbonyl protecting group (optionallysubstituted), an arylthiocarbonyl protecting group (optionallysubstituted), and combinations thereof), wherein, optionally, the RNAhas at least one phosphorus protecting group (e.g., substituted andunsubstituted: alkyl, benzyl, alkylbenzyl, dialkylbenzyl,trialkylbenzyl, thioalkylbenzyl, phenylthiobenzyl, dithioalkylbenzyl,trithioalkylbenzyl, thioalkylhalobenzyl, alkyloxybenzyl,dialkyloxybenzyl, halobenzyl, dihalobenzyl, trihalobenzyl, esterifiedsalicyl, alkylnitrile protecting groups, as well as other protectinggroups described herein); deprotecting (e.g., deprotecting,simultaneously or independently, one or more of 2′-hydroxyl protectinggroup, the base protecting group, and/or the phosphorus protectinggroup) the RNA in a solution including an α-effect nucleophile (e.g.,hydrogen peroxide, peracids, perboric acids, alkylperoxides, hydrogenperoxide salts, hydroperoxides, butylhydroperoxide, benzylhydroperoxide,phenylhydroperoxide, performic acid, peracetic acid, perbenzoic acid,chloroperbenzoic acid and all corresponding salts of these species,mixtures with other compounds (e.g., sodium formate) and combinationsthereof), wherein the solution is at a pH of about 4 to 11 (e.g, a pH ofabout 6 to 11, and a pH of about 8 to 11), and wherein the α-effectnucleophile has a pKa of about 4 to 13; (prior to or after deprotecting)optionally, cleaving (e.g., simultaneously or independently) the RNAfrom the support; and optionally, precipitating the RNA out of thesolution.

In another embodiment of the present disclosure, exemplary methods ofdeprotecting polynucleotides, among others, includes: synthesizing on asolid support a ribonucleic acid (RNA), wherein the RNA has at least one2′-hydroxyl protecting group and at least one protected exocyclic amineon a heterocyclic base; wherein said 2′-hydroxyl group is protected withan orthoester protective group; introducing the RNA to a solutionincluding an α-effect nucleophile, wherein the solution is at a pH ofabout 6 to 11 and wherein the α-effect nucleophile has a pKa of about 4to 13; deprotecting said 2′-orthoester protecting group under acidicconditions; (prior to or after cleaving) optionally, simultaneously orindependently cleaving the RNA from the support; and optionally,precipitating the RNA out of the solution.

In another embodiment of the present disclosure, exemplary methods ofdeprotecting polynucleotides, among others, includes: synthesizing on asolid support a ribonucleic acid (RNA), wherein the RNA has at least one2′-hydroxyl protecting group and at least one protected exocyclic amineon a heterocyclic base; wherein said 2′-hydroxyl protecting groupincludes a acetal protective group; introducing the RNA to a solutionincluding an α-effect nucleophile, wherein the solution is at a pH ofabout 6 to 11 and wherein the α-effect nucleophile has a pKa of about 4to 13, deprotecting said 2′-acetal protecting group under acidicconditions; (prior to or after cleaving) optionally, simultaneously orindependently cleaving the RNA from the support; and optionally,precipitating the RNA out of the solution.

In another embodiment of the present disclosure, exemplary methods ofdeprotecting polynucleotides, among others, includes: synthesizing on asolid support a ribonucleic acid (RNA), wherein the RNA has at least one2′-hydroxyl protecting group and at least one protected exocyclic amineon a heterocyclic base; wherein said 2′-hydroxyl protecting group istrisiopropyloxymethyl (TOM) group; introducing the RNA to a solutionincluding an α-effect nucleophile, wherein the solution is at a pH ofabout 6 to 11 and wherein the α-effect nucleophile has a pKa of about 4to 13; subsequently, deprotecting said 2′-trisiopropyloxymethylprotecting group under fluoride anions conditions; optionally, cleavingthe RNA from the support; and optionally, precipitating the RNA out ofthe solution.

In another embodiment of the present disclosure, exemplary methods ofdeprotecting polynucleotides, among others, includes: synthesizing on asolid support a ribonucleic acid (RNA), wherein the RNA has at least one2′-hydroxyl protecting group and at least one protected exocyclic amineon a heterocyclic base; wherein said 2′-hydroxyl protecting groupincludes a tert-butyldimethylsilyl (TBDMS) protective group; introducingthe RNA to a solution including an α-effect nucleophile, wherein thesolution is at a pH of about 6 to 11 and wherein the α-effectnucleophile has a pKa of about 4 to 13; deprotecting said 2′-TBDMSprotective group with fluoride anions; (prior to or after cleaving)optionally, simultaneously or independently cleaving the RNA from thesupport; and optionally, precipitating the RNA out of the solution.

In another embodiment of the present disclosure, exemplary methods ofdeprotecting polynucleotides, among others, include: synthesizing on apolystyrene solid support a ribonucleic acid (RNA), wherein the RNA hasat least one phosphorus protecting group, at least one 2′-hydroxylprotecting group and at least one protected exocyclic amine on aheterocyclic base; wherein said phosphorus protecting group is a methyland said 2′-hydroxyl protecting group is trisiopropyloxymethyl (TOM)group; deprotecting said methyl group with thiophenol or derivative ofthiophenol; then deprotecting said 2′-trisiopropyloxymethyl protectinggroup under fluoride anions conditions; subsequently, introducing theRNA to a solution including an α-effect nucleophile, wherein thesolution is at a pH of about 6 to 11 and wherein the α-effectnucleophile has a pKa of about 4 to 13; deprotecting the excocyclicamine protecting group; optionally, simultaneously or independentlycleaving the RNA from the support; and optionally precipitating the RNAout of the solution.

In another embodiment of the present disclosure, exemplary methods ofdeprotecting polynucleotides, among others, include: synthesizing on apolystyrene solid support a ribonucleic acid (RNA), wherein the RNA hasat least one phosphorus protecting group, at least one 2′-hydroxylprotecting group and at least one protected exocyclic amine on aheterocyclic base; wherein said phosphorus protecting group is a methyland said 2′-hydroxyl protecting group is a tert-butyldimethylsilyl(TBDMS) protective group; deprotecting said methyl group with thiophenolor derivative of thiophenol; then deprotecting said 2′-TBDMS protectinggroup under fluoride anions conditions; subsequently, introducing theRNA to a solution including an α-effect nucleophile, wherein thesolution is at a pH of about 6 to 11 and wherein the α-effectnucleophile has a pKa of about 4 to 13; deprotecting said exocyclicamine protective group; optionally, simultalneously or independentlycleaving the RNA from the support; and optionally precipitating the RNAout of the solution.

In another embodiment of the present disclosure, exemplary methods ofdeprotecting polynucleotides, among others, includes: synthesizingoptionally on a solid support a polynucleotide wherein thepolynucleotide has at least one protected exocyclic amine on aheterocyclic base and at least one protected imine on a heterocyclicbase; deprotecting the exocyclic amino groups by introducing thepolynucleotide to a solution including an α-effect nucleophile, whereinthe solution is at a pH of about 6 to 11, and wherein the α-effectnucleophile has a pKa of about 4 to 13; optionally, simultaneously orindependently cleaving the polynucleotide from the support; andoptionally, precipitating the polynucleotide out of the solution.

In another embodiment of the present disclosure, exemplary methods ofdeprotecting polynucleotides, among others, includes: synthesizingoptionally on a solid support a polynucleotide wherein thepolynucleotide has at least one protected exocyclic amine on aheterocyclic base, at least one protected imine on a heterocyclic baseand at least one 2′-hydroxyl protecting group; deprotecting theexocyclic amino groups by introducing the polynucleotide to a solutionincluding an α-effect nucleophile, and wherein the solution is at a pHof about 6 to 11 wherein the α-effect nucleophile has a pKa of about 4to 13; optionally, simultaneously or independently cleaving thepolynucleotide from the support; and optionally, precipitating thepolynucleotide out of the solution.

In another embodiment of the present disclosure, exemplary methods ofdeprotecting polynucleotides, among others, includes: synthesizingoptionally on a solid support a polynucleotide wherein thepolynucleotide has at least one protected exocyclic amine on aheterocyclic base, at least one protected imine on a heterocyclic baseand at least one 2′-hydroxyl protecting group; deprotecting theexocyclic amino groups and the 2′-hydroxyl protecting groups byintroducing the polynucleotide to a solution including an α-effectnucleophile, wherein the solution is at a pH of about 6 to 11 andwherein the α-effect nucleophile has a pKa of about 4 to 13; optionally,simultaneously or independently cleaving the polynucleotide from thesupport; and optionally, precipitating the polynucleotide out of thesolution.

2′-Hydroxyl Protecting Groups and Deprotection

Embodiments of the disclosure include nucleotide monomers andpolynucleotide compounds including at least one nucleotide moiety unit,where the nucleotide monomer and the nucleotide moiety each include a2′-hydroxyl protecting group and in some embodiments additionalprotecting groups. The nucleotide monomer and the nucleotide moiety aredifferent, in at least one way, in that the nucleotide moiety is part ofa polynucleotide compound, while the nucleotide monomer is not part of apolynucleotide compound. The nucleotide moiety includes a linking moietythat links a plurality of nucleotide moieties together (and/or to asubstrate), while the nucleotide monomer does not include such a linkingmoiety. For the following discussion the structures will be referred toas “nucleotide monomer”, but it is to be understood that the structurescan be nucleotide moieties if a linking group is included in thestructure to interconnect nucleotide moieties in a polynucleotidecompound.

As mentioned above, embodiments of the disclosure can include nucleotidemonomers having a 2′-hydroxyl protecting group. A variety of 2′-hydroxylprotecting groups have been used for RNA synthesis. The 2′-hydroxylprotecting groups can be used in conjunction with other protectinggroups (e.g., base protecting groups and/or phosphorus protectinggroups). In an embodiment of the present disclosure, the 2′-hydroxylprotecting groups can include, but are not limited to, groups that canbe removed with peroxyanions as well as groups that have beenspecifically developed and optimized to be removed by peroxyanions. Inaddition, the 2′-hydroxyl protecting groups can include, but are notlimited to, the structures (e.g., structure I) described in furtherdetail below.

Exemplary 2′-hydroxyl protecting groups include, but are not limited to:acid-labile protecting groups, nucleophile-labile protecting groups,fluoride-labile protecting groups, and oxidative-labile protectinggroups. These groups are attached to the 2′-oxygen through a variety offunctionalities that include, but are not limited to, esters,carbonates, thiocarbonates, carbamates, acetals, thioacetals, carbonatesof hemiacetals, carbonates of thiohemiacetals, orthoesters,thioorthoesters, carbonates of orthoesters, carbonates ofthioorthoesters, silyl, siloxanes, carbonates and thiocarbonates ofsilanes, carbonates and thiocarbonates of siloxanes, silyl protectedhemiacetals, carbonates and thiocarbonates of silyl protectedhemiacetals, carbonates and thiocarbonates of hemiacetals, carbonatesand thiocarbonates of thiohemiacetals, and a variety of substitutedderivatives of any of the previously described functionalities.

The nucleotide monomer can include, but is not limited to, a structuresuch as structures I. The following structure illustrates embodimentsthat include 2′-hydroxyl protecting groups.

B can include, but is not limited to, a base and a base including aprotection group.

In addition, the nucleotide monomer can include, but is not limited to,a structure such as structures II. The following structure illustratesembodiments that include 2′-hydroxyl protecting groups.

For structure I and II, R₁ and R₂ are each independently selected from agroup such as, but not limited to, H, a protecting group, and

wherein only one of R₁ and R₂ is

In an embodiment, R₁ and/or R₂ can include a linking moiety thatinterconnects the nucleotide moieties in a polynucleotide or connects toa substrate.

R₃ is a group such as, but not limited to, an alkyl group, an arylgroup, a substituted alkyl, and a substituted aryl group. In anembodiment, R3 can include a linking moiety that interconnects thenucleotide moieties in a polynucleotide or connects to a substrate.

R₄ and R₅ are each independently selected from a group such as, but notlimited to, an alkyl group, an aryl group, a substituted alkyl, and asubstituted aryl group. In addition, R₄ and R₅ can be, optionally,cyclically connected to each other. In an embodiment, R₄ and/or R₅ caninclude a linking moiety that interconnects the nucleotide moieties in apolynucleotide.

In an embodiment, R1 and R2 may each individually be selected from oneof the following: H, a protecting group, and:

but where both R1 and R2 are not

R₃ can include, but is not limited to, an alkyl group, a substitutedalkyl group, an aryl group, and a substituted aryl group. In anembodiment, R₃ is optionally a linking moiety that links to anothernucleotide moiety of a polynucleotide or connects to a substrate. Nuc isa nucleotide or polynucleotide.

In another embodiment, R1 and R2 can have the following structure:

R′ and R″ are each individually a group such as, but not limited to, H,an alkyl group, an aryl group, a substituted alkyl, a substituted arylgroup. In an embodiment, R′ and/or R″ can include a linking moiety thatinterconnects the nucleotide moieties in a polynucleotide or connects toa substrate.

R₃ is a group such as, but not limited to, an alkyl group, an arylgroup, a substituted alkyl, and a substituted aryl group. In anembodiment, R3 can include a linking moiety that interconnects thenucleotide moieties in a polynucleotide or connects to a substrate.

R₄ and R₅ are each independently selected from a group such as, but notlimited to, an alkyl group, an aryl group, a substituted alkyl, and asubstituted aryl group. In addition, R₄ and R₅ can be, optionally,cyclically connected to each other. In an embodiment, R₄ and/or R₅ caninclude a linking moiety that interconnects the nucleotide moieties in apolynucleotide or connects to a substrate.

2HPG is a 2′-hydroxyl protecting group such as, but not limited to, oneof the groups listed below I, II, III, IVa, IVb, V, VI, VIIa, VIb, andVIII:

Group I:

R₆, R₇, and R₈ are each independently selected from a group such as, butnot limited to, an alkyl group, a substituted alkyl, a substituted alkylwith an electron-withdrawing group, a substituted aryl group with anelectron-withdrawing group, a group that is converted into an electronwithdrawing group by oxidation (e.g., via peroxyanions), an aryl group,a substituted aryl group, where R₆, R₇, and R₈ are not each a methylgroup, where R₆, R₇, and R₈ are not each an unsubstituted alkyl, andwhere R₆, R₇, and R₈ are optionally cyclically connected to each other.

Group II:

R₆, R₇, and R₈ are each independently selected from a group such as, butnot limited to, an alkyl group, an aryl group, a substituted alkylgroup, and a substituted aryl group, and R₆, R₇, and R₈ are optionallycyclically connected to each other.

Group III:

R₉ is a group such as, but not limited to, an alkyl group, an arylgroup, a substituted alkyl with an electron-withdrawing group, and asubstituted aryl group with an electron-withdrawing group, where R₁₀ andR′₁₀ are each independently selected from a group such as, but notlimited to, H, an alkyl group, and a substituted alkyl with anelectron-withdrawing group.

It should be noted that bonds (e.g., indicated by lines) that aredirected into the center of a ring structure (e.g., benzene ring) meanthat the bond can be to any one of the carbons of the ring that are onlybonded to a hydrogen and another carbon of the ring. SG can include oneor more groups, where each group is attached to one of the carbons inthe carbon ring. SG is a group such as, but not limited to, H, an alkylgroup, an aryl group, a substituted alkyl with an electron-donatinggroup, and a substituted aryl group with an electron-donating group.

Group IVa and IVb:

R₆ and R₇ are independently selected from a group such as, but notlimited to, an alkyl group, an aryl group, a substituted alkyl with anelectron-withdrawing group, and a substituted aryl group with anelectron-withdrawing group, R₆ and R₇ are optionally cyclicallyconnected to each other or to the phenol and where R₁₁ is a group suchas, but not limited to, an ester protecting group and silyl protectinggroup.

It should be noted that bonds (e.g., indicated by lines) that aredirected into the center of a ring structure (e.g., benzene ring) meanthat the bond can be to any one of the carbons of the ring that are onlybonded to a hydrogen and another carbon of the ring. R₁₂ can include oneor more groups, where each group is attached to one of the carbons inthe carbon ring. R₁₂ is a group such as, but not limited to, an alkylgroup, an aryl group, a substituted alkyl with an electron-withdrawinggroup, and a substituted aryl group with an electron-withdrawing group.

Group V:

R₆ and R₇ are independently selected from a group such as, but notlimited to, an alkyl group, an aryl group, a substituted alkyl with anelectron-withdrawing group, and a substituted aryl group with anelectron-withdrawing group, where R₆ and R₇ are optionally cyclicallyconnected to each other and where R₁₃ is an amide-protecting group.

It should be noted that bonds (e.g., indicated by lines) that aredirected into the center of a ring structure (e.g., benzene ring) meanthat the bond can be to any one of the carbons of the ring that are onlybonded to a hydrogen and another carbon of the ring. R₁₂ can include oneor more groups, where each group is attached to one of the carbons inthe carbon ring. R₁₂ is a group such as, but not limited to, an alkylgroup, an aryl group, a substituted alkyl with an electron-withdrawinggroup, and a substituted aryl group with an electron-withdrawing group.

Group VI:

R₆ and R₇ are independently selected from a group such as, but notlimited to, an alkyl group, an aryl group, a substituted alkyl with anelectron-withdrawing group, and a substituted aryl group with anelectron-withdrawing group, and R₆ and R₇ are optionally cyclicallyconnected to each other.

It should be noted that bonds (e.g., indicated by lines) that aredirected into the center of a ring structure (e.g., benzene ring) meanthat the bond can be to any one of the carbons of the ring that are onlybonded to a hydrogen and another carbon of the ring. SG and SR₁₄ caneach include one or more groups, where each group is attached to one ofthe carbons in the carbon ring. SR₁₄ is one or more thioetherindependently selected from a thioalkyl group, a thioaryl group, asubstituted thioalkyl group, and a substituted thioaryl group. In anembodiment, R₁₄ includes, but is not limited to, an alkyl group, an arylgroup, a substituted alkyl group, and a substituted aryl group. SG is agroup such as, but not limited to, H, an alkyl group, an aryl group, asubstituted alkyl with an electron-donating group, a substituted arylgroup with an electron-donating group, and an electron-donating group inthe ortho or para position to the thioether SR₁₄.

Groups VIIa and VIIb:

R₆ and R₇ are independently selected from a group such as, but notlimited to, an alkyl group, an aryl group, a substituted alkyl with anelectron-withdrawing group, and a substituted aryl group with anelectron-withdrawing group, where R₆ and R₇ are optionally cyclicallyconnected to each other or to the phenol and where R₁₁ is a group suchas, but not limited to, an ester protecting group and silyl protectinggroup.

It should be noted that bonds (e.g., indicated by lines) that aredirected into the center of a ring structure (e.g., benzene ring) meanthat the bond can be to any one of the carbons of the ring that are onlybonded to a hydrogen and another carbon of the ring. R₁₂ can include oneor more groups, where each group is attached to one of the carbons inthe carbon ring. R₁₂ is a group such as, but not limited to, an alkylgroup, an aryl group, a substituted alkyl with an electron-withdrawinggroup, and a substituted aryl group with an electron-withdrawing group.

Group VIII:

R₆ and R₇ are independently selected from a group such as, but notlimited to, an alkyl group, an aryl group, a substituted alkyl with anelectron-withdrawing group, and a substituted aryl group with anelectron-withdrawing group, where and R₆ and R₇ are optionallycyclically connected to each other.

It should be noted that bonds (e.g., indicated by lines) that aredirected into the center of a ring structure (e.g., benzene ring) meanthat the bond can be to any one of the carbons of the ring that are onlybonded to a hydrogen and another carbon of the ring. SG and SR₁₄ caneach include one or more groups, where each group is attached to one ofthe carbons in the carbon ring. SR₁₄ is one or more thioethersindependently selected from a thioalkyl group, a thioaryl group, asubstituted thioalkyl group, and a substituted thioaryl group. In anembodiment R₁₄ can include, but is not limited to, an alkyl group, anaryl group, a substituted alkyl group, and a substituted aryl group. SGis a group such as, but not limited to, H, an alkyl group, an arylgroup, a substituted alkyl with an electron-donating group, asubstituted aryl group with an electron-donating group, and anelectron-donating group in the ortho or para position to the thioetherSR₁₄.

It should be noted that some properties may be considered when selectinga 2′-hydroxyl protecting group and these include, but are not limitedto, whether the groups has specificity for introduction on the2′-hydroxyl (regioselectivity), whether the group does not lead toisomerisation products, whether the group does not compromise couplingyield, and whether the group can be easily removed remove withoutdegrading the RNA.

Regioselective Introduction

The 2′-hydroxyl protecting groups can be placed on the molecule ineither a regioselective manner, a non-regioselective manner, or aregiospecific manner. In a regioselective manner, the protecting groupis setup by various conditions and circumstances in the chemicalreaction to react only with the hydroxyl region that is desired, in thiscase the 2′-hydroxyl.

An example of a regioselective protecting group is the dimethoxytritylprotecting group [Schaller, H.; Weimann, G.; Lorch, B.; Khorana, H. G.,J Am. Chem. Soc. 1963, 85, 3821-3827, which is incorporated herein byreference]. The dimethoxytrityl protecting groups will regioselectivelyreact with only the 5′-hydroxyl. This protecting group is regioselectivebecause the rate of reaction of dimethoxytrityl chloride with the5′-hydroxyl is at least 100 times faster than with either the 2′ or 3′hydroxyls. The 2′-hydroxyl can then be regioselectively ornon-regioselectively reacted with the desired 2′-protective group. In aregioselective manner, the desired 2′-hydroxyl protecting group istypically reacted with 5′-protected nucleosides using various asymmetricLewis Acid catalysts. Although it is possible to find conditions andreagents that give some level of regioselectivity, completeregioselectivity is difficult to achieve using this method, and thisapproach tends to produce a mixture of structural isomers. The desired2′-protected nucleoside must then be separated from the undesired3′-protected nucleoside or the bis-2′, 3′-protected nucleosidechromatographically. Typically this approach is used to enrich theamount of the desired 2′-protected nucleoside produced in order todecrease the cost of obtaining the correct 2′-protected nucleoside.Rarely has it been demonstrated that this approach achieves completeregioselectivity. Often differential regioselectivity is demonstratedfor the different heterobase containing nucleosides. Completeregioselectivity may be obtained with one or more nucleosides, butusually one or more of the nucleosides show little or noregioselectivity resulting, in low synthetic yield of the desiredmonomer.

It is therefore most often preferable to utilize a regiospecificapproach to protection of the 2′-hydroxyl. In a regiospecific manner anucleoside, that can contain a heterobase protecting group, is typicallythen reacted on the 5′ and 3′ hydroxyls with an additional blockinggroup that transiently protects both positions simultaneously. Anexample of such a blocking group is the 1,3-tetraisopropyl disiloxane[Markiewicz W. T., J Chem Research (S) 1979 24-25)]. The desired2′-hydroxyl protecting group is then reacted specifically with the 240-hydroxyl.

This is usually the most preferred method of protecting of the2′-hydroxyl. However, there are several examples whereby the desired2′-hydroxyl protecting group is incompatible with the blocking groupsneeded to transiently protect the 3′ and 5′ hydroxyls. As an example,the 1,3-tetraisopropyl disiloxane is a transient blocking group that isused to block the 5′ and 3′ hydroxyls simultaneously, allowing the2′-hydroxyl to then be regioselectively reacted. The 1,3-tetraisopropyldisiloxane group is subsequently removed using a solution of fluorideions. Due to the required deprotection conditions of the1,3-tetraisopropyl disiloxane group, 2′-protecting groups that containsilyl or silicon atoms are not compatible with this regioselectiveapproach [Beigelman, L, and Serebryany, V, Nucleosides, Nucleotides, andNucleic Acids, 2003, Vol 22, 1007-1009, which is incorporated herein byreference].Isomerization

Additionally, the stability of the 2′-hydroxyl protecting group tomigration is an important factor that limits the scope and theusefulness of a variety of protecting group classes or species within aclass of protecting group. Because the 2′-hydroxyl moiety is “cis” tothe 3′-hydroxyl it can promote the isomerization of many classes ofprotecting groups from the 2′-hydroxyl to the 3′-hydroxyl and viseversa. Often this isomerization reaction is catalyzed by the addition ofweak acids or weak bases. This becomes problematic for silica gelpurification, since silica gel is weakly acidic and therefore protectinggroups that are susceptible to acid catalyzed isomerization aredifficult to purify by column chromatography. A similar reaction canoccur during formation of the 3′-phosphoramidite. There are two basicmethods for the formation of phosphoramidites. The first is through theuse of a chlorophosphine [Beaucage, S. L.; Caruthers, M. H. TetrahedronLett. 1981, 22, 1859-1862, which is incorporated herein by reference].The use of a chlorophosphine reagent requires exposure of the nucleosideto a weak base like triethyl amine or diisopropylethyl amine. Protectinggroups that are susceptible to base catalyzed isomerization aredifficult to phosphitylate by this method as shown below.

Alternatively, phosphoramidites can be prepared by the“bis-dialkylaminophosphine” method [Barone A D, Tang J Y, Caruthers M H,Nucleic Acids Res., 1984, 12(10), 4051-61, which is incorporated hereinby reference]. This method uses a weak acid such as tetrazole of theamine salt of tetrazole to catalyze the reaction of thebis-dialkylaminophosphine with the 3′-hydroxyl.

Protecting groups that undergo isomerization under these conditions canbe difficult to phosphitylate forming the 3′-phosphoramidite. It istherefore preferable to utilize protecting group classes or specieswithin a protecting group that are not susceptible to isomerizationunder either weak acid or weak base conditions. A clear example of aprotecting group that can isomerize under weak acid and weak baseconditions is the silyl TBDMS. Although unidentified by the authors, theoriginal method published for the use of this protecting group for the2′-hydroxyl [Usman N, Ogilvie K K, Jiang M Y, Cedergren R J, J Am. Chem.Soc., 1987, 109(25), 7845-7854] gave only isomerized mixtures of thephosphoramidite monomers and as a result it was necessary to develophighly specialized techniques for preparation and purification to limitthe amount of isomerized products in the phosphoramidite monomers.Another protecting group class that is particularly susceptible toisomerize is the ester class. The isomerization of esters can beinhibited by the use of large bulky alkyl or aryl groups. Thereforelarge bulky esters are particularly preferred as 2′-protecting groupsover smaller groups like acetyl.

Carbonates and Thiocarbonates:

Carbonates and thiocarbonates do not undergo isomerization from the2′hydroxyl to the 3′ hydroxyl and vice versa. The chemical transitionthat would lead to isomerization always results in the formation of 2′,3′-cyclic carbonates, which are the thermodynamically favored product.The formation of 2′, 3′-cyclic carbonates under acidic or basicconditions can be quite facile and lead to low yields of the desired2′-protected product.

However, tertiary carbonates as shown by Losse and tertiarythiocarbonates as discovered in the present disclosure are resistant toformation of 2′-3′ cyclic carbonates and are particularly useful as2′-protection [Losse G, Suptitz G, Krusche, K, Journal für PraktischeChemie, 1992, 334(6), 531-532, which is incorporated herein byreference].

Carbonates are particularly useful since, even if the cyclic carbonateis formed at some percentage during the synthesis of the monomers, thecyclic carbonate does not produce a reactive monomer (3′-isomer) withthe protecting group on the 3′-hydroxyl. This is especially important toensure only formation of 5′-3′-linked RNA molecules. However, alltertiary carbonates are not useful for the synthesis of RNA molecules.Tertiary alkylcarbonates can be quite stable to nucleophiles andresistant to cleavage by peroxyanions. The tertiary butyloxycarbonylgroup was previously described for the protection of the 2′-hydroxyl forsolid-phase RNA synthesis. The resistance of this group to removal by avariety of mild pH conditions demonstrated the unsuitability of thisgroup for RNA synthesis [Losse G, Naumann, W, Winkler, A, Suptitz G,Journal für Praktische Chemie, 1994, 336(3), 233-236, which isincorporated herein by reference]. In order to remove this protectinggroup the authors needed to employ a high concentration (4 molar) ofhydrochloric acid in dioxane. It has also been demonstrated that thisprotecting group was quite stable to the mildly basic but highlynucleophillic conditions of peroxyanion cleavage using 2 molar hydrogenperoxide at pH 9.5. Therefore, it was found that the carbonate moietyshould be modified to make the group more labile to nucleophiles andspecifically to peroxyanion nucleophiles. This can be accomplished in atleast two methods. The first is the addition of electron withdrawinggroups to the alkyl or aryl substituents that can then affect thestability of the cleavage products formed as shown below.

An example of adding an electron-withdrawing group to a tertiarycarbonate includes the example of using one or more trifluoromethylgroups to replace one or more of the methyl groups on the tertiarybutyloxycarbonyl group as shown below.

A particularly useful method of adding an electron-withdrawing group toa tertiary carbonate (see below) is to replace one or more of the methylgroups on the tertiary butyloxycarbonyl group with a substituted benzenering and to have the benzene ring substituted with one or more electronwithdrawing groups.

Electron-withdrawing groups can either exist as part of the protectinggroup prior to cleavage by peroxyanions, or be created by thedeprotection reaction. A particularly useful approach illustrated belowis to use substituents on the alkyl or aryl groups that areelectron-donating and to have those substituents chemically converted toelectron-withdrawing groups in the solution of peroxyanion. The electrondonating effect helps stabilize the protecting group during theoligomerization process, and then this effect is reversed duringcleavage of the protecting group to produce an electron-withdrawinggroup that stabilizes the products from the cleavage reaction and thenaids the facile removal under mild pH conditions.

An example of adding an electron-withdrawing group to a tertiarycarbonate is the example of using one or more thiomethyl ethers toreplace one or more of the methyl groups on the tertiarybutyloxycarbonyl group. Thiomethylethers are typically stable tooxidation in the presence of the dilute iodine solutions that are usedfor oxidation of the phosphite triester to phosphate triester during theroutine phosphoramidite DNA or RNA synthesis cycle. However, in thepresence of dilute solutions of hydroperoxides, peracids, orcombinations of hydroperoxides and peracids, the thiomethylether israpidly oxidized to the corresponding sulfone (as shown below). Themethyl sulfone is a strongly electron-withdrawing group that stabilizesthe products from the cleavage reaction and then aids the facile removalof the under mild pH conditions.

A particularly convenient method of adding a moiety to a tertiarycarbonate that can then be chemically converted to anelectron-withdrawing group by peroxyanions is the direct substitution ofa sulfur atom for an oxygen atom in the bridging position to form athiocarbonate (see below).

From data on the cleavage and oxidation of thiocarbonates, it appearsthat the sulfur is not oxidized prior to the cleavage. Tertiarybutylthiocarbonyl protected nucleosides exposed to 1 to 2 molarsolutions of hydrogen peroxide at pH 6.0 do not generate sulfuroxidation products or cleavage of the thiocarbonate group, even after a24-hour exposure. However, if the pH is raised to 9.5, the cleavage ofthe thiocarbonate is group is complete in approximately 10 minutes. Thedata suggests that once the cleavage reaction occurs, using anucleophillic solution of hydrogen peroxide, the resulting mercaptan israpidly oxidized to the sulfonate, making the reaction rapid andirreversible.

The second method for making the group more labile to nucleophiles, andspecifically to peroxyanion nucleophiles, is to incorporate a moiety ormoieties that enhances removal of the protecting group by afragmentation process that creates thermodynamically stabile fragmentsillustrated below. This stabilizes the products of the cleavage reactionpromoting facile removal of protecting groups under mild pH conditions.

Examples of such protecting groups are the tertiary carbonates ofthiohemiacetals. Shown below is an example of 2′-hydroxyl protectionusing 2-thiomethylpropane-2-oxycarbonyl. Upon cleavage by peroxyanionnucleophiles the protecting group is fragmented into the highlythermodynamically stable products of acetone and methanesulfonate.

Oxidizable 2′-Hydorxyl Protecting Groups

Another embodiment includes nucleotide monomers and polynucleotides thatinclude nucleotide moieties, where each of the nucleotide monomers andthe nucleotide moieties include, but are not limited to, a protectinggroup (e.g., group I, group II, , group VI, group VIIa, group VIIb,group VIII,) that can undergo oxidative transformations to enhance thelability of the protecting group towards the nucleophilic removal. Theoxidative transformation can occur prior to the cleavage, typicallygenerating an electron withdrawing species on the protecting group thatdid not exist prior to the oxidation reaction or after the cleavagereaction to generate a species that cannot participate in an equilibriumreaction to reform the protecting group. An example of a species thatundergoes an oxidative transformation to produce an electron withdrawingspecies that makes the protecting group more labile is the2-methylthiobenzoyl group. The 2-methylthiobenzoyl group has similarstability to benzoyl or 2-methylbenzoyl. However, upon exposure to abuffered 6% hydrogen peroxide solution at pH 9.5, the 2-methylthiomoiety is oxidized to a methylsulfone, and the methylsulfone derivativeis significantly more labile to nucleophiles than benzoyl or2-methylbenzoyl. The resulting 2-methylsulfonbenzoyl is cleaved rapidlyby the hydroperoxide anion. An example of an oxidative transformationthat occurs after the cleavage of the protecting group is thet-butylthiocarbonate. The sulfur atom on the t-butylthiocarbonate is notoxidized when exposed to 6% hydrogen peroxide at pH 5.0, and itslability towards nucleophile is similar to t-butylcarbonate. However,using 6% hydrogen peroxide at pH 9.5, rapidly generates thet-butylsulfonic acid and the removal of the protecting group is quitefacile. This is in stark contrast to the use of t-butyl carbonate as a2′-hydroxyl protecting group, which was shown to be convenient tointroduce onto the 2′-hydroxyl of a nucleoside and resistant to theformation of the 2′-3′-cyclic carbonate. However difficult, if notimpossible, to remove this protecting group without destroying thedesired RNA products. The t-butyl carbonate was shown to be veryresistant to nucleophiles and was removed using 5 molar solutions ofhydrochloric acid. The present disclosure includes polynucleotide (e.g.,RNA) that include combinations of nucleotide moieties such as, but notlimited to, compounds of structure I having one or more protectinggroups as described herein, compounds of structure II having one or moreprotecting groups as described herein, nucleotide moities having one ormore protecting groups as described herein, nucleotide moieties notincluding protecting groups, nucleotide ,moieties known in the art,combinations thereof, and the like. In an embodiment, the presentdisclosure includes a polynucleotide compound having structure III:

where B can be, but is not limited to, a base including a protectiongroup; where R₃ can be a group such as, but not limited to, an alkylgroup, an aryl group, a substituted alkyl, and a substituted aryl group;and where 2HPG is a 2′-hydroxyl protecting group selected from groups Ithrough VIII. It should be noted that the structure above could bebonded to one or more nucleotide moieties and/or to a substrate. In anembodiment, the present disclosure includes a polynucleotide compoundhaving structure IV:

where B can be, but is not limited to, a base and a base includingprotecting group, wherein each protecting group is selected from one ofthe following: an exocyclic amino protection group and an iminoprotecting group; where R₃ can be a group such as, but not limited to,an alkyl group, an aryl group, a substituted alkyl, a substituted arylgroup; and where 2HPG is identical for both the 2′-hydroxyl protectinggroup and the heterobase exocyclic amino protected group selected fromgroups I through VIII. It should be noted that the structure above couldbe bonded to one or more nucleotide moieties and/or to a substrate.Heterocyclic Base Protecting Groups

Embodiments of the disclosure include nucleotide monomers andpolynucleotide compounds including at least one nucleotide moiety unit,where the nucleotide monomer and the nucleotide moiety each include aheterocyclic base protecting group. The nucleotide monomer and thenucleotide moiety are different, in at least one way, in that thenucleotide moiety is part of a polynucleotide compound, while thenucleotide monomer is not part of a polynucleotide compound. Thenucleotide moiety includes a linking moiety that links a plurality ofnucleotide moieties, while the nucleotide monomer does not include sucha linking moiety. For the following discussion the structures may bereferred to as “nucleotide monomer”, but it is to be understood that thestructures can be nucleotide moieties if a linking group is included inthe structure to interconnect nucleotide moieties in a polynucleotidecompound.

Exocyclic amines of the aglycone need to be protected duringpolynucleotide synthesis. Typically, the following exocyclic amines N-4of cytidine, N-6 of adenosine and N-2 of guanosine require, protectionduring RNA synthesis. Sometimes, the imino group can require additionalprotection. In the case of imino protection, the N-3 or O-4 of uridineand the N-1 or O-6 of guanosine can require a protecting group. In mostcases, the protecting groups utilized for exocyclic amines can also beapplied to the protection of the imino group through a screeningprocess.

Embodiments of the disclosure can include nucleotide monomers andpolynucleotide compounds (e.g., ribonucleotide compounds) includingnucleotide monomers and moieties having 2′-hydroxyl protecting groups asdescribed herein as well as heterocyclic base protecting groups. Thepolynucleotide can include nucleotide monomers and moieties havingstructures XX through XXII. Embodiments of the position of the baseprotecting groups (APG) are shown on structures XX through XXIII:

The APGs can include, but are not limited to, base protecting groupsthat are removed under a same set of conditions as the 2HPG (e.g., inperoxyanions solutions). For example, the APG and the 2HPG can beremoved in a single step when exposed to a peroxyanion solution. Forexample, APG can be acetyl, chloroacetyl, dicholoracetyl,trichloroacetyl, fluoroacetyl, difluoroacetyl, trifluoroacetyl,nitroacetyl, propionyl, n-butyryl, i-butyryl, n-pentanoyl, i-pentanoyl,t-pentanoyl, phenoxyacteyl, 2-chlorophenoxyacetyl,t-butyl-phenoxyacetyl, methylthioacetyl, phenylthioacetyl,2-chlorophenylthioacetyl, 3-chlorophenylthioacetyl,4-chlorophenylthioacetyl, t-butyl-phenylthioacetyl, benzoyl,2-nitrobenzoyl, 3-nitrobenzoyl, 4-nitrobenzoyl, 2-chlorobenzoyl,3-chlorobenzoyl, 4-chlorobenzoyl, 2,4-di-chlorobenzoyl, 2-fluorobenzoyl,3-fluorobenzoyl, 4-fluorobenzoyl, 2-trifluoromethylbenzoyl,3-trifluoromethylbenzoyl, 4-trifluoromethylbenzoyl, benzyloxycarbonyl,and the like.

The APGs can include, but are not limited to, base protecting groupsthat are removed under a different set of conditions as the 2HPG. Thesegroups that can not be removed by peroxyanion depend strongly on thebase (A, G, or C) they are protecting. Most of the amidine protectinggroups such as dimethylformamidine, dibutylformamidine,dimethylacetamidine, diethylacetamidine are stable to peroxyanions.

R1 and R2 are each individually selected from one of the following: H, aprotecting group, and

but R1 and R2 are both not:

R1 and R2 can also be a linking moiety that interconnects a plurality ofnucleotide moieties in a polynucleotide.

R₃ can include, but is not limited to, an alkyl group, a substitutedalkyl group, an aryl group, and a substituted aryl group. R₃ canoptionally be a linking moiety that links to another nucleotide moietyof a polynucleotide or connected to a substrate. R₄ and R₅ can eachindependently include, but are not limited to, an alkyl group, asubstituted alkyl group, an aryl group, a substituted aryl group, acyclic alkyl, a substituted cyclic alkyl, a heterocycle, a substitutedheterocycle, an aryl group, and a substituted aryl group and, optionallya linking moiety that links to another nucleotide moiety of thepolynucleotide or connected to a substrate.

X can include, but is not limited to, H, OH, halogen, an alkoxy group, asubstituted alkoxy group, an aryloxy group, a substituted aryloxy group,an amino group, a substituted amino group, a cyano group, an azidogroup, a sulfonic acid group, a protecting group, and an O-protectinggroup.

The APGs can include, but are not limited to, exocyclic amino protectinggroups. In addition, the APG can include, but are not limited to, baseprotecting groups that are removed under a same set of conditions as the2′-hydroxyl protecting groups (e.g., in peroxyanions solutions). Forexample, the APG and the 2′-hydroxyl protecting groups can be removed ina single step when exposed to a peroxyanion solution.

In an embodiment, the APG is a moiety that can undergo oxidativetransformations in peroxyanion solutions (as discussed in more detailbelow) to enhance the lability of the protecting group towardsnucleophillic removal. These oxidative transformations can occur priorto the cleavage, typically generating an electron withdrawing species onthe protecting group that did not exist prior to the oxidation reactionor after the cleavage reaction to generate a species that cannotparticipate in an equilibrium reaction to reform the protecting group.

The 2-methylthiobenzoyl group is an example of a species that undergoesan oxidative transformation to produce and electron withdrawing speciesthat makes the protecting group more labile. The 2-methylthiobenzoylgroup has similar stability to benzoyl or 2-methylbenzoyl. However, uponexposure to a buffered 6% hydrogen peroxide solution at about pH 9.5,the 2-methylthio moiety is oxidized to a methylsulfone, and themethylsulfone derivative is significantly more labile to nucleophilesthan benzoyl or 2-methylbenzoyl. The resulting 2-methylsulfonbenzoyl iscleaved rapidly by the hydroperoxide anion.

The t-butylthiocarbamate group is an example of an oxidativetransformation that occurs after the cleavage of the protecting group.The sulfur atom on the t-butylthiocarbamate is not oxidized when exposedto 6% hydrogen peroxide at pH 5.0, and the lability to nucleophile issimilar to t-butylcarbamate. However, using 6% hydrogen peroxide atabout pH 9.5 rapidly renders the t-butylsulfonic acid, and the removalof the protecting group quite facile.

It should also be noted that as for the protection of heterobaseprotecting groups, a particularly useful set of the 2′-hydroxylprotecting group includes a moiety that can undergo oxidativetransformations in peroxyanion solutions to enhance the lability of theprotecting group towards nucleophillic removal, or a moiety that can becleaved in peroxyanion solutions thus be deprotected simultaneously withthe heterobase protecting groups in one single step (one pot reaction).These 2′-hydroxyl protecting groups include, but are not limited to: anester protecting group, a carbonate protecting group, a thiocarbonateprotecting group, and a carbamate protecting group.

The APGs can include, but are not limited to, base protecting groupssuch as those having the following structures:

It should be noted that Q is an atom such as, but not limited to, sulfur(S) and oxygen (O), and that R is a group such as, but not limited to,an alkyl group, a substituted alkyl group, an aryl group, and asubstituted aryl group. R′ and R″ are each independently selected from agroup such as, but not limited to, a halogen, an alkyl group, asubstituted alkyl group, an aryl group, and a substituted aryl group.R₁₅ is a group such as, but not limited to, an alkyl group, an arylgroup, a substituted alkyl, and a substituted aryl group.

It should be noted that bonds (e.g., indicated by lines) that aredirected into the center of a ring structure (e.g., benzene ring) meanthat the bond can be to any one of the carbons of the ring that are onlybonded to a hydrogen and another carbon of the ring. R₁₆ can include oneor more groups, where each group is attached to one of the carbons inthe carbon ring. Each R₁₆ is independently a group such as, but notlimited to, H, a halogen, a hydroxyl group, an alkoxy group, asubstituted alkoxy group, an aryloxy group, a substituted aryloxy group,an amino group, a substituted amino group, a nitro group, a nitrilegroup, an alkyl group, an aryl group, a substituted alkyl, and asubstituted aryl group. In an embodiment, when there are more than oneR₁₆ (e.g., multiple R₁₆'s bonded to different carbons on the carbonring), then two or more of R₁₆ are optionally cyclically connected toeach other. R₁₇, R₁₈, and R₁₉, are each independently a group such as,but not limited to, H, an alkyl group, an aryl group, a substitutedalkyl, and a substituted aryl group. In an embodiment, two or three ofR₁₇, R₁₈, and R₁₉ can be cyclically connected to each other.

In another embodiment, the nucleotide polymer can include, but is notlimited to, a nucleotide polymer that includes at least one structuresuch as those shown in structures I through III above. Embodiments ofthe position of the base protecting groups (APG) on the bases are shownon structures XX through XXII above.

It should be noted that in this embodiment that R1 and R2 are eachindividually be selected from one of the following: H, a protectinggroup, and:

but where both R1 and R2 are not

R₃ can include, but is not limited to, an alkyl group, a substitutedalkyl group, an aryl group, a substituted aryl group, and, optionally, alinking moiety that links to another nucleotide moiety of apolynucleotide. Nuc is a nucleotide or polynucleotide.

The APGs can include, but are not limited to, base protecting groupsthat are removed under a different set of conditions than the2′-hydroxyl protecting groups, for example wherein the 2′-hydroxylprotecting groups include, but are not limited to, TBDMS, TOM, ACE,acetals such as THP, and derivatives of acetals (Ctmp and the like).

In addition, the APGs can include, but are not limited to, baseprotecting groups such as acetyl, chloroacetyl, dicholoracetyl,trichloroacetyl, fluoroacetyl, difluoroacetyl, trifluoroacetyl,nitroacetyl, propionyl, n-butyryl, i-butyryl, n-pentanoyl, i-pentanoyl,t-pentanoyl, phenoxyacteyl, 2-chlorophenoxyacetyl,t-butyl-phenoxyacetyl, methylthioacetyl, phenylthioacetyl,2-chlorophenylthioacetyl, 3-chlorophenylthioacetyl,4-chlorophenylthioacetyl, t-butyl-phenylthioacetyl, benzyloxycarbonyl,(9-fluoroenyl)-methoxycarbonyl (Fmoc), 2-nitrophenylsulfenyl,4-nitrophenylethylcarbonyl, 4-nitrophenylethoxycarbonyl,diphenylcarbamoyl, morpholinocarbamoyl, dialkylformamidines, succinyl,phthaloyl, benzoyl, 4-trifluoromethylbenzoyl, 2-methylbenzoyl,3-methylbenzoyl, 4-methylbenzoyl, 2, 4-dimethylbenzoyl, 2,6-dimethylbenzoyl, 2,4, 6-trimethylbenzoyl, 2-methoxybenzoyl,3-methoxybenzoyl, 4-methoxybenzoyl, 2, 4-dimethoxybenzoyl, 2,6-dimethoxybenzoyl, 2, 4, 6-trimethoxybenzoyl, 2-methylthiobenzoyl,3-methylthiobenzoyl, 4-methylthiobenzoyl, 2, 4-dimethylthiobenzoyl, 2,6-dimethylthiobenzoyl, 2, 4, 6-trimethylthiobenzoyl, 2-chlorobenzoyl,3-chlorobenzoyl, 4-chlorobenzoyl, 2, 4-dichlorobenzoyl, 2,6-dicholorbenzoyl, 2-fluorobenzoyl, 3-fluorobenzoyl, 4-fluorobenzoyl, 2,4-difluorobenzoyl, 2, 6-difluorobenzoyl, 2, 5-difluorobenzoyl, 3,5-difluorobenzoyl, 2-trifluoromethylbenzoyl, 3-trifluoromethylbenzoyl,2, 4-trifluoromethylbenzoyl, 2, 6-trifluoromethylbenzoyl, 2,5-trifluoromethylbenzoyl, 3, 5-trifluoromethylbenzoyl, 2-nitrobenzoyl,3-nitrobenzoyl, 4-nitrobenzoyl, (3-methoxy 4-phenoxy)benzoyl,(triphenyl)silylethyleneoxycarbonyl,(diphenylmethyl)silylethyleneoxycarbonyl,(phenyldimethyl)silylethyleneoxycarbonyl,(trimethyl)silylethyleneoxycarbonyl,(triphenyl)silyl(2,2-dimethyl)ethyleneoxycarbonyl,(diphenylmethyl)silyl[(2,2-dimethyl)ethylene]oxycarbonyl,phenyldimethylsilyl[(2,2-dimethyl)ethylene]oxycarbonyl,trimethylsilyl[(2,2-dimethyl)ethylene]oxycarbonyl, methyloxycarbonyl,ethyloxycarbonyl, propyloxycarbonyl, isopropyloxycarbonyl,butyloxycarbonyl, isobutyloxycarbonyl, t-butyloxycarbonyl,phenyloxycarbonyl, benzyloxycarbonyl, methylthiocarbonyl,ethylthiocarbonyl, propylthiocarbonyl, isopropylthiocarbonyl,butylthiocarbonyl, isobutylthiocarbonyl, t-butylthiocarbonyl,phenylthiocarbonyl, benzylthiocarbonyl, methyloxymethyleneoxycarbonyl,methylthiomethylene-oxycarbonyl, phenyloxymethylene-oxycarbonyl,phenylthiomethyleneoxycarbonyl, methyloxy(methyl)methyleneoxycarbonyl,methylthio(methyl)methyleneoxycarbonyl,methyloxy(dimethyl)methyleneoxycarbonyl,methylthio(dimethyl)methyleneoxycarbonyl,phenyloxy(methyl)methyleneoxycarbonyl,phenylthio(methyl)methyleneoxycarbonyl,phenyloxy(dimethyl)methyleneoxycarbonyl,phenylthio(dimethyl)methyleneoxycarbonyl, and substituted derivatives ofany of these previously described groups.

It should also be noted that as for the protection of the 2′-hydroxylgroup, a particularly useful set of heterobase protecting groups is amoiety that can undergo oxidative transformations in peroxyanionsolutions (as discussed in more detail below) to enhance the lability ofthe protecting group towards nucleophilic removal. These oxidativetransformations can occur prior to the cleavage, typically generating anelectron withdrawing species on the protecting group that did not existprior to the oxidation reaction or after the cleavage reaction togenerate a species that cannot participate in an equilibrium reaction toreform the protecting group. An example of a species that undergoes anoxidative transformation to produce an electron withdrawing species thatmakes the protecting group more labile is the 2-methylthiobenzoyl group.The 2-methylthiobenzoyl group has similar stability to benzoyl or2-methylbenzoyl. However, upon exposure to a buffered 6% hydrogenperoxide solution at pH 9.5, the 2-methylthio moiety is oxidized to amethylsulfone, and the methylsulfone derivative is significantly morelabile to nucleophiles than benzoyl or 2-methylbenzoyl. The resulting2-methylsulfonbenzoyl is cleaved rapidly by the hydroperoxide anion. Anexample of an oxidative transformation that occurs after the cleavage ofthe protecting group is the t-butylthiocarbamate. The sulfur atom on thet-butylthiocarbamate is not oxidized when exposed to 6% hydrogenperoxide at pH 5.0, and the lability to nucleophiles is similar tot-butylcarbamate. However using 6% hydrogen peroxide at a pH of about9.5, rapidly generates the t-butylsulfonic acid and the removal of theprotecting group is quite facile.

In another embodiment, R1 and R2 can have the following structure:

R′ and R″ are each individually a group such as, but not limited to, H,an alkyl group, an aryl group, a substituted alkyl, and a substitutedaryl group. In an embodiment, R′ and/or R″ can include a linking moietythat interconnects the nucleotide moieties in a polynucleotide orconnected to a substrate.

R₃ is a group such as, but not limited to, an alkyl group, an arylgroup, a substituted alkyl, and a substituted aryl group. In anembodiment, R3 can include a linking moiety that interconnects thenucleotide moieties in a polynucleotide or connected to a substrate.

R₄ and R₅ are each independently a group such as, but not limited to, analkyl group, an aryl group, a substituted alkyl, and a substituted arylgroup. In addition, R₄ and R₅ can be, optionally, cyclically connectedto each other. In an embodiment, R₄ and/or R₅ can include a linkingmoiety that interconnects the nucleotide moieties in a polynucleotide orconnected to a substrate.

In an embodiment, the APGs can include, but are not limited to, baseprotecting groups that are removed under a same set of conditions as the2HPG (e.g., in peroxyanions solutions). For example, the APG and the2HPG can be removed in a single step when exposed to a peroxyanionsolution. For example, APG can be acetyl, chloroacetyl, dicholoracetyl,trichloroacetyl, fluoroacetyl, difluoroacetyl, trifluoroacetyl,nitroacetyl, propionyl, n-butyryl, i-butyryl, n-pentanoyl, i-pentanoyl,t-pentanoyl, phenoxyacteyl, 2-chlorophenoxyacetyl,t-butyl-phenoxyacetyl, methylthioacetyl, phenylthioacetyl,2-chlorophenylthioacetyl, 3-chlorophenylthioacetyl,4-chlorophenylthioacetyl, t-butyl-phenylthioacetyl, benzoyl,2-nitrobenzoyl, 3-nitrobenzoyl, 4-nitrobenzoyl, 2-chlorobenzoyl,3-chlorobenzoyl, 4-chlorobenzoyl, 2,4-di-chlorobenzoyl, 2-fluorobenzoyl,3-fluorobenzoyl, 4-fluorobenzoyl, 2-trifluoromethylbenzoyl,3-trifluoromethylbenzoyl, 4-trifluoromethylbenzoyl, benzyloxycarbonyland the like.

In an embodiment, the APGs can include, but are not limited to, baseprotecting groups that are removed under a different set of conditionsas the 2HPG. These groups that can not be removed by peroxyanion dependstrongly on the base (A, G or C) they are protecting. Most of theamidine protecting groups such as dimethylformamidine,dibutylformamidine, dimethylacetamidine, diethylacetamidine are stableto peroxyanions.

Deprotecting

As mentioned above, embodiments of the present disclosure includemethods for deprotecting one or more protecting groups of apolynucleotide (e.g., a RNA) molecule such as those described herein. Inparticular, the methods include deprotecting a RNA molecule in asolution of an α-effect nucleophile (e.g., a peroxyanion solution),where the α-effect nucleophile has a pKa of about 4 to 13. In addition,the solution is at a pH of about 6 to 11.

One advantage of using a mildly basic solution including an α-effectnucleophile is that the solution including an α-effect nucleophile iscompatible with standard phosphoramidite methods for polynucleotidesynthesis. Further, the deprotected RNA molecules are stable and showlittle or no degradation for an extended period of time when stored inthe solution including the a-effect nucleophile. An additional advantageof using the solution including an α-effect nucleophile is that thedisadvantages associated with the 3- and 2-step processes typically usedfor RNA deprotecting are not present.

It should be noted that embodiments of the present disclosure includemethods that are multi-step methods and use the α-effect nucleophile(e.g., for deprotection of the one or more 2′-hydroxyl protectinggroups, one or more base protecting groups, and/or one or morephosphorus protecting groups) may be one of a number of steps used inthe synthesis and/or deprotection of the polynucleotide. For example,one step of the method may use the α-effect nucleophile to deprotect oneor more protecting groups, while one or more other steps may includeother solutions (e.g., TBDMS, TOM, ACE, and like chemistries) used todeprotect one or more other protecting groups.

In general, the methods involve forming and/or providing a synthetic RNAmolecule, where the RNA molecule has at least one of the following: abase having a protecting group, a 2′-hydroxyl protecting group, aphosphorus protecting group, and combinations thereof. Then, the RNAmolecule is mixed with a solution including a least one type of anα-effect nucleophile, where the solution is at a pH of about 4 to 11. Inaddition, the α-effect nucleophile has a pKa of about 4 to 12. One ormore of the protecting groups can be deprotected through interactionwith the α-effect nucleophile.

In general, these solutions including the α-effect nucleophiles can bepredominately buffered aqueous solutions or buffered aqueous/organicsolutions. Under these conditions it is convenient and cost effective toremove the deprotecting solutions by simple precipitation of the desiredRNA oligonucleotides directly from the deprotecting mixture by additionof ethanol to the solution. Under these conditions, the RNA is pelletedto the bottom of a centrifuge tube and the deprotecting mixturecontaining the α-effect nucleophile is removed by simply pouring off thesupernatant and rinsing the pellet with fresh ethanol. The desired RNAis then isolated by resuspending in a typical buffer for chromatographicpurification or direct usage in the biological experiment of interest.Because of the nature of most α-effect nucleophiles, removal of thedeprotection reagents from the desired RNA products is significantlyless tedious and time consuming; this is especially true in comparisonto the use of a fluoride-ion solution for final deprotection of the RNAmolecule.

One significant advantage for post synthetic deprotection applied to anymethod of RNA synthesis is that the α-effect nucleophile solution can beexploited to remove a variety of commonly used protecting groups, theprotecting groups described herein, or linkers under pH conditions thatdo not catalyze rapid degradation of RNA. Unlike the commonly applieduse of strong bases and/or typical nucleophiles for post syntheticdeprotection of RNA, partial loss of the 2′-protecting group, prior toor during exposure to the α-effect nucleophiles, does not result incleavage of the internucleotide bond. Therefore, the use of the α-effectnucleophile solutions simply for the deprotection of heterobase blockinggroups has significant advantages over current methods even if coupledwith the use of routine protecting groups. These advantages become evenmore significant if they are used with protecting groups describedherein that are specifically optimized for the rapid removal underoxidative, nucleophilic conditions at neutral to mildly basic pH.

The solution of an α-effect nucleophile typically may have a pH in therange of about 4 to 11, about 5 to 11, about 6 to 11, about 7 to 11,about 8 to 11, about 4 to 10, about 5 to 10, about 6 to 10, about 7 to10, or about 8 to 10. In particular embodiments, the solution has a pHof about 7 to 10. It should also be noted that the pH is dependent, atleast in part, upon the α-effect nucleophile in the solution and theprotecting groups of the RNA. Appropriate adjustments to the pH can bemade to the solution to accommodate the α-effect nucleophile.

The α-effect nucleophiles can include, but are not limited to,peroxyanions, hydroxylamine derivatives, hydroximic acid and derivativesthererof, hydroxamic acid and derivatives thereof, hydrazine andderivatives thereof, carbazide and derivatives thereof, semicarbazidesand derivatives thereof, and combinations thereof. The peroxyanionα-effect nucleophiles can include compounds such as, but not limited to,hydrogen peroxide and salts thereof, peracids and salts thereof,perboric acids and salts thereof, alkylperoxides and salts thereof,hydroperoxides and salts thereof, butylhydroperoxide and salts thereof,benzylhydroperoxide and salts thereof, phenylhydroperoxide and saltsthereof, performic acid and salts thereof, peracetic acid and saltsthereof, perbenzoic acid and salts thereof, chloroperbenzoic acid andsalts thereof, benzoic acids and salts thereof, substituted perbenzoicacids and salts thereof, cumene hydroperoxide and salts thereof,perbutric acid and salts thereof, tertriarylbutylperoxybenzoic acid andsalts thereof, decanediperoxoic acid and salts thereof, and combinationsthereof.

Hydrogen peroxide, salts of hydrogen peroxide, and mixtures of hydrogenperoxide and performic acid are especially useful. Hydrogen peroxide,which has a pKa around 11, is particularly useful for deprotectingsolutions above pH 9.0. Below pH 9.0 there is generally no sufficientconcentration of peroxyanion to work as an effective nucleophile. BelowpH 9.0 it is especially useful to use mixtures of hydrogen peroxide andperacids. These peracids can be preformed and added to the solution orthey can be formed in situ by the reaction of hydrogen peroxide and thecarboxylic acid or carboxylic acid salt.

An example is that an equal molar mixture of hydrogen peroxide andsodium formate can be used at pH conditions below 9.0 as an effectiveperoxyanion deprotecting solution where hydrogen peroxide alone is notan effective deprotecting mixture. The utility of peracids tends to bedependent upon the pKa of the acid and size of molecule: the higher thepKa of the acid, the more useful as a peroxyanion solution, and thelarger the size of the molecule, the less useful. Typically the pKa ofthe peracid is lower than the pH of the desired peroxyanion solution.

The α-effect nucleophiles typically used in these reactions aretypically strong oxidants, therefore the concentration of the reagent inthe deprotecting solution should be limited in order to avoid oxidativeside products where undesired. The α-effect nucleophiles are typicallyless than about 30% weight/vol of the solution, more typically betweenabout 0.1% and 10% weight/vol of the solution and most typically about3% to 7% weight/vol of the solution. The typical 3% solution of hydrogenperoxide is about 1 molar hydrogen peroxide. A solution of between 1molar and 2 molar hydrogen peroxide is especially useful. A typicalsolution of hydrogen peroxide and performic acid is an equal molarmixture of hydrogen peroxide and performic acid, both in the range ofabout 1 to 2 molar. An example of an in situ prepared solution ofperformic acid is about 2 molar hydrogen peroxide and about 2 molarsodium formate buffered at about pH 8.5.

In typical embodiments, the α-effect nucleophile is characterized ashaving a pKa in the range from about 4 to 13, about 4 to 12, about 4 to11, about 5 to 13, about 5 to 12, about 5 to 11, about 6 to 13, about 6to 12, about 6 to 11, about 7 to 13, about 7 to 12, or about 7 to 11.

It should also be noted that the pKa is a physical constant that ischaracteristic of the specific α-effect nucleophile. Chemicalsubstitution and solvolysis conditions can be used to raise or lower thepKa and therefore specifically optimize the conditions of deprotecting.Appropriate selection of the α-effect nucleophile should be madeconsidering the other conditions of the method and the protecting groupsof the RNA. In addition, mixtures of peroxides and hydroperoxides can beused with molecules to form peroxyanions in situ.

As an example a solution of hydrogen peroxide can be used with asolution of formic acid at pH conditions below 9.0. At pH conditionsless than 9.0, hydrogen peroxide is not significantly ionized due to itsionization constant of around 11. At pH 7.0, only about 0.01% of thehydrogen peroxide is in the ionized form of the α-effect nucleophile.However, the hydrogen peroxide can react in situ with the formic acid toform performic acid in a stable equilibrium. At pH 7.0, the performicacid is significantly in the ionized form and is an active α-effectnucleophile. In general, the lower the pKa, the more ionized; the higherthe pKa, the better the nucleophile. The advantage of such an approachis that solutions of performic acid tend to degrade rapidly andstabilizers need to be added. The equilibrium that is formed between thehydrogen peroxide solutions and the formic acid helps stabilize theperformic acid such that it can be used to completely deprotect the RNAprior to degrading. Performic acid is especially useful in a bufferedmixture of hydrogen peroxide at pH 8.5 because the pKa of performic acidis approximately 7.1. Peracetic acid is useful at pH 8.5 but less usefulthan performic acid because the pKa of peracetic acid is approximately8.2. At pH 8.5, peracetic acid is only about 50% anionic, whereas at pH8.5 performic acid is more than 90% anionic.

In general, the pKa for the hydroperoxides is about 8 to 13. The pKa forhydrogen peroxide is quoted to be about 10 to 12, depending upon themethod of analysis and solvent conditions. The pKa for thealkylperoxides is about 8 to 14. The pKa for the peracids is about 3 to9. In embodiments where the peroxyanion is hydroperoxide, the solutionis at pH of about 9 to 11. In embodiments where the peroxyanion ishydrogen peroxide, the solution is at pH of about 9 to 10.

In embodiments where the peroxyanion is an alkylperoxide, the solutionis at pH of about 8 to 11. In embodiments where the peroxyanion is aperacid, the solution is at pH of about 6 to 9. In addition, the peracidhas a pKa of about 4 to 10.

In addition, the aqueous buffer solution includes a buffer such as, butnot limited to, tris(hydroxymethyl)aminomethane, aminomethylpropanol,citric acid, N,N′-bis(2-hydroxyethyl)glycine,2-[bis(2-hydroxyethyl)amino]-2-(hydroxy-methyl)-1,3-propanediol,2-(cyclohexylamino)ethane-2-sulfonic acid,N-2-hydroxyethyl)piperazine-N′-2-ethane sulfonic acid,N-(2-hydroxyethyl)piperazine-N′-3-propane sulfonic acid,morpholinoethane sulfonic acid, morpholinopropane sulfonic acid,piperazine-N,N′-bis(2-ethane sulfonic acid),N-tris(hydroxymethyl)methyl-3-aminopropane sulfonic acid,N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid,N-tris(hydroxymethyl)methylglycine, and combinations thereof.

Substrates for Solid Phase Synthesis

The polynucleotides (one or more units) can be attached to suitablesubstrates that may have a variety of forms and compositions. Thesubstrates may derive from naturally occurring materials, naturallyoccurring materials that have been synthetically modified, or syntheticmaterials. Examples of suitable support materials include, but are notlimited to, nitrocellulose, glasses, silicas, teflons, and metals (e.g.,gold, platinum, and the like). Suitable materials also include polymericmaterials, including plastics (for example, polytetrafluoroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike), polysaccharides such as agarose (e.g., that availablecommercially as Sepharose®, from Pharmacia) and dextran (e.g., thoseavailable commercially under the tradenames Sephadex® and Sephacyl®,also from Pharmacia), polyacrylamides, polystyrenes, polyvinyl alcohols,copolymers of hydroxyethyl methacrylate and methyl methacrylate, and thelike.

One advantage of using α-effect nucleophiles is that unlike methodsusing fluoride-based solutions for the final deprotection of RNA, theα-effect nucleophiles are compatible with either polymeric or silicasubstrates. In this regard, silica substrates can be used since they areless expensive than polymeric substrates.

In contrast, fluoride ion final deprotection conditions attack thesilica substrate in a similar manner as attacks the silicon protectinggroups it is attempting to remove. As a result, the silica substrate canbe dissolved or partially dissolved, giving a significant amount offluorosilicate impurities that are difficult to remove. Also, the attackon the substrate decreases the effective concentration of the fluoridereagent used for deprotection, requiring longer deprotection times andhigher concentrations of fluoride reagent. As a result, the use offluoride ion final deprotection is often limited to the use of polymericsupports or multiple step final deprotections.

While the foregoing embodiments have been set forth in considerabledetail for the purpose of making a complete disclosure, it will beapparent to those of skill in the art that numerous changes may be madeto such details without departing from the spirit and the principles ofthe disclosure. Accordingly, the disclosure should be limited only bythe following claims.

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties.

EXAMPLES

FIGS. 2A through 2E illustrate chromatographs of a synthetic RNA (SEQ.ID NO. 1, 5′ GUCACCAGCCCACUUGAG 3′) and a solution of 5% hydrogenperoxide in a solution having a pH of about 8 at various times (FIG. 2A(time_(RNA)=0), FIG. 2B (time_(HP)=0), FIG. 2C (time=3 hours), FIG. 2D(time=12 hours), and FIG. 2E (time=24 hours)).

The synthetic RNA showed little or no degradation in a solution of 5%hydrogen peroxide in a solution having a pH of about 8 for up to 24hours.

General Examples Transient Protection of Hydroxyl Moieties

The chemical protection of the exocyclic amino groups on the heterobasesof DNA and RNA are typically accomplished using the “Jones procedures”(FIG. 3) that were initially described by Ti et. al. J. Am. Chem Soc1982, 104, 1316-1319, which was incorporated herein by reference. Inthis procedure a nucleoside was transiently protected using excesstrimethylsilyl chloride in pyridine. The trimethylsilyl groups reactwith all available hydroxyls and amines on the molecule. Thetrimethylsilyl group blocks the reaction of the hydroxyls with anyfurther protecting groups, but the exocyclic amine remains reactive eventhough they can contain trimethylsilyl groups. This procedure can beadapted and optimized by anyone skilled in the art to a variety ofsubstrates. For example, a larger excess of trimethylsilyl chloride wasuseful for obtaining higher yields on guanosine nucleosides (Fan el.al., Org. Lett. Vol 6. No. 15, 2004). If the nucleoside substratealready contains protective groups on one or more of the hydroxylresidues, the excess of trimethylsilyl chloride was scaled back. Thisprocedure can also be used for protection of the imino groups onguanosine, thymidine and uridine.

Once the nucleoside has been transiently protected with trimethylsilylgroups, the exocyclic amine groups can be reacted with any number ofamino reactive protecting groups. Examples of these, for illustrationnot exclusion, are acid chlorides, active esters such as p-nitrophenylester, chloroformates, thiochloroformates, acid anhydrides,pyrocarbonates, dithiopyrocarbonates, and the like.

Transient Protection of 5′ and 3′ Hydroxyl Moieties For RegioselectiveProtection of the 2′-Hydroxyl.

The chemical protection of the 5′ and 3′ hydroxyl moiety of a nucleosidewas typically accomplished using a disiloxane deravitive known astetraisopropyldisiloxane dichloride (TIPS). This protecting groupsimultaneously blocks the 5′ and 3′ hydroxyls to allow for completeregioselective introduction of a protective group on to the 2′-hydroxyl(FIG. 4) [Markiewicz, W. T., J. Chem Research (S) 1979 24-25) which wasincorporated herein by reference].

Preparation of Novel Chloroformates as Amino and Hydroxyl ReactiveProtecting Groups

The preparation chloroformates includes using solutions of phosgene.Alcohols and mercaptans are typically reacted with excess phosgene atdry ice temperatures. It was often important to add one equivalent of anon-nucleophillic base, like pyridine or triethyl amine, to bothcatalyze the reaction and neutralize the HCl formed. The order ofaddition should be considered since, during the reaction, it wassignificant that the phosgene was generally in high concentrationrelative to the alcohol or mercaptan; this prevents the formation ofcarbonates or thiocarbonates. The phosgene solution (6 molarequivalents) was typically cooled on a dry ice/ethanol bath, and analcohol or mercaptan solution in toluene/pyridine was added dropwise.The solution was allowed to warm to room temperature and was filteredunder a blanket of dry argon gas. The resulting clear solution wasevaporated to an oil using a rotary evaporator attached to a Teflon headdiaphragm pump. The excess phosgene can be removed by evaporation, sincephosgene was a gas at room temperature. The evaporation process removesthe solvent and excess phosgene. The exhaust from the pump was bubbledthrough an aqueous solution of KOH to neutralize the excess phosgene.The temperature was controlled during the evaporation since somechloroformates can have low boiling points. Tertiary chloroformates aretypically made using metal salts of alcohols and mercaptans. In thiscase, the metal salt, such as sodium salt, was formed on the alcohol ormercaptan prior to reacting with phosgene, and typically anon-nucleophilic base was not required.

FIG. 5 illustrates the selective protection of exocyclic amine withchloroformate reagents. FIG. 6 illustrates the selective protection of2′-hydroxyl with chloroformate reagents.

Simultaneous Protection of Amino and Hydroxyl Moieties with NovelProtecting Groups

Nucleosides can be protected using the Markiewicz procedure to give5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl) ribonucleosides. Thosemonomers can then be reacted with either chloroformates orpyrocarbonates to produce ribonucleosides simultaneously protected withthe same protecting group. This procedure has the specific advantage ofstreamlining the synthesis of nucleoside monomers for RNA synthesis andthus reducing the cost and complexity of synthesis. The ability toutilize the same protecting group on both the heterobase and 2′-hydroxylwas specifically enabled through the use of peroxyanions for the finaldeprotection. Peroxyanion nucleophillic cleavage at mildly basic pHallows for the deprotection of both groups under pH conditions that doesnot give rise to cyclization and cleavage of the intemucleotide bond. Iftypical nucleophiles were used to simultaneously remove the protectivegroups from the exocyclic amine and 2′-hydroxyl it would require thatthe reactions occur under strongly basic conditions. Under the stronglybasic conditions of typical nucleophiles, removal of the 2′-hydroxylprotective group would immediately result in cyclization and cleavage ofthe internucleotide bond.

FIG. 7 illustrates the preparation of5′,3′-O-(tTetraisopropyldisiloxane-1,3-diyl) ribonucleosides. FIG. 8illustrates the simultaneous protection of5′,3′-O-(tetraisopropyldisiloxane-1,3-diyl) ribonucleosides usingchloroformates or pyrocarbonates.

Preparation of Pyrocarbonates as Amino and Hydroxyl Reactive ProtectingGroups

Many tertiary chloroformates are only stable at low temperatures.However, most tertiary thiochloroformates are stable at roomtemperature. The instability of tertiary chloroformates at roomtemperature makes it difficult to isolate and use these reagents. As aresult, pyrocarbonates were selected for use in the preparation ofnucleoside N-carbonyloxy compounds that have a tertiary carbon attachedto the oxygen. These pyrocarbonates are significantly more stable atroom temperature than the corresponding chloroformates. Pyrocarbonatesare typically made using the metal salt or trimethylsilyl derivatives oftertiary alcohols. The metal salt of the alcohol was reacted in anon-polar solvent, such as hexanes with carbon dioxide, to form thecorresponding carbonic acid. One half of one equivalent of methanesulfonyl chloride was added to the reaction to form the pyrocarbonate.The reaction was then quenched with a 5% aqueous solution of sulfuricacid and the pyrocarbonate isolated by evaporation of the hexanes layer.

FIG. 9 illustrates the selective protection of exocyclic amine withpyrocarbonate reagents. FIG. 10 illustrates the selective protection of2′-hydroxyl with pyrocarbonate reagents.

General Procedure for the Synthesis ofO-Trimethylsilylhemimethylthioacetals as Intermediate in the Preprationof Chloroformate Amino Reactive Protecting Groups (Group IV)

Preparation of KCN-18-Crown-6 Complex

The potassium cyanide-18-crown-6 complex was prepared by the dissolutionof 1 equivalent of potassium cyanide in anhydrous methanol of 1equivalent of 18-crown-6 (Aldrich). The solvent was removed at 65° C.using a Teflon head diaphragm pump, followed by drying under high vacuumat room temperature for 15 to 20 min.

A dry, round bottom flask was charged with 1 equivalent of the aldehydeand 1.1 equivalent of an alkyl or arylthiotrimethylsilane. Upon additionof 5×10⁻⁴ equivalents of the solid potassium cyanide-18-crown-6 complex,the reaction was initiated. Often the reaction becomes exothermic andrequires cooling with an ice bath. Upon completion of the reaction, theO-trimethylsilylhemimethylthioacetal products were typically isolated bydirect distillation from the crude mixture.

FIG. 11 illustrates the selective protection of exocyclic amine withhemimethylthioacetal chloroformate reagents. FIG. 12 illustrates thesynthesis of O-trimethylsilylhemimethylthioacetals as intermediates inthe preparation of the corresponding chloroformate.

General Procedure for the Synthesis ofO-Trimethylsilylhemimethylthioketals as Intermediates in the Preparationof Pyrocarbonates for the Synthesis of Group V Amino Reactive ProtectingGroups

To a dry 25-mL flask was added 10 mg (0.03 mmol)) of anhydrous zinciodine, 10 mg (0.15 mmol) of imidazole, and 10 mmol of the aldehyde orketone in 5 mL of anhydrous ether. To this stirred solution was added 22mmol of the appropriate thiosilane. General reaction time of 1 hr at 25°C. was observed. Typically, the products were isolated by distillationafter dilution with ether, followed by extraction with water. The etherlayer was typically evaporated, and the residual distilled at reducedpressure.

FIG. 13 illustrates O-trimethylsilylhemimethylthioketals as intermediatein the preparation of the corresponding pyrocarbonate. FIG. 14illustrates the selective protection of exocyclic amine withhemimethylthioketal pyrocarbonate reagents. FIG. 15 illustrates theselective protection of 2′-hydroxyl with hemimethylthioketalpyrocarbonate reagents.

Exocyclic Amino Protecting Group Examples Group I Synthesis ofN-(methylthiomethyloxycarbonyl) Ribonucleosides

Acetic acid methylthiomethyl ester (50 mmol) was purchased from TCIAmerica (Portland, OR) and dissolved in 200 mL of ether, and 100 mL of a1.0 M solution of KOH in water was added. The reaction was allowed tostir overnight and the ether solution separated and evaporated to anoil. The resulting methylthiomethyl hemiacetal was dissolved inanhydrous toluene with an equal molar amount of anhydrous pyridine. Aphosgene solution (6 molar equivalents) was cooled on a dry ice/ethanolbath, and the hemiacetal solution added dropwise. The solution wasallowed to warm to room temperature and filtered under a blanket of dryargon gas. The resulting clear solution was evaporated to an oil using arotary evaporator attached to a Teflon head diaphragm pump to producemethylthiomethylchloroformate. The evaporation process removed thesolvent and excess phosgene. The exhaust from the pump was bubbledthrough an aqueous solution of KOH to neutralize the excess phosgene. Aribonucleoside (10 mmole) was coevaporated 3 times with pyridine andthen dried on vacuum pump for 2 hours. Anhydrous pyridine (50 mL) andtrimethylsilyl chloride (8.8 ml, 70 mmole) were added, and the mixturewas stirred at room temperature for 2 hours.Methylthiomethylchloroformate (20 mmole) was then added, and stirringcontinued for another 12 hours. Water (10 mL) was added to quench thereaction and hydrolyze trimethylsilyl groups. The reaction mixture wasleft overnight. Crude product was evaporated to remove the excesspyridine, and 200 mL of DCM was added with 5% aqueous solution ofNaHCO₃. The precipitated product was dried and utilized in the nextreactions.

Group II Synthesis of N-(methylthiocarbamate) Ribonucleosides

Methylthiochloroformate was purchased from Aldrich. A ribonucleoside (10mmole) was coevaporated 3 times with pyridine, and then dried on avacuum pump for 2 hours. Anhydrous pyridine (50 mL) and trimethylsilylchloride (8.8 ml, 70 mmole) were added, and the mixture was stirred atroom temperature for 2 hours.

Methylthiochloroformate (20 mmole) was then added, and stirringcontinued for another 12 hours. Water (10 mL) was added to quench thereaction and hydrolyze trimethylsilyl groups. The reaction mixture wasleft overnight. Crude product was evaporated to remove the excesspyridine, and 200 mL of DCM was added with 5% aqueous solution ofNaHCO₃. The precipitated product was dried and utilized in the nextreactions.

Group III Synthesis of 5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl)N-thiomethylacetyl Riboguanosine

5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl) riboguanosine (20 mmole) wascoevaporated 3 times with pyridine and then dried on vacuum pump for 2hours. Anhydrous pyridine (100 mL) and trimethylsilyl chloride (12.8 ml,100 mmole) were added, and the mixture was stirred at room temperaturefor 2 hours. Thiomethyacetyl chloride (24 mmole) was then added, andstirring continued for another 12 hours. Water (100 mL) was added toquench the reaction and hydrolyze trimethylsilyl groups. The reactionmixture was left for 1 hour. Crude product was extracted with DCM,washed with 5% aqueous solution of NaHCO₃, and purified by columnchromatography using CHCl₃ with a gradient of methanol (0-3%). The yieldwas about 69%.

Group IV Synthesis of N-(carbonyloxy-1-phethylthiomethyl-1-H-isobutane)Ribonucleosides

A dry, round bottom flask was charged with 5 mL (55.1 mmol) ofisobutyraldehyde and 10.4 g (57 mmol) of phenylthiosilane. Upon additionof 10 mg of the solid potassium cyanide-18-crown-6 complex, the reactionwas initiated. The reaction became exothermic and required cooling withan ice bath. Upon completion of the reaction, theO-trimethylsilyl-1-phethylthiomethyl-1-H-isobutane, 11.3 grams (81%yield), was isolated by direct distillation from the crude mixture at71° C. at 0.05 mm Hg.

The resulting O-trimethylsilyl-1-phethylthiomethyl-1-H-isobutane wasdissolved in anhydrous toluene with an equal molar amount of anhydrouspyridine. A phosgene solution (6 molar equivalents) was cooled on a dryice/ethanol bath, and theO-trimethylsilyl-1-phethylthiomethyl-1-H-isobutane solution addeddropwise. The solution was allowed to warm to room temperature andfiltered under a blanket of dry argon gas. The resulting clear solutionwas evaporated to an oil using a rotary evaporator attached to a Teflonhead diaphragm pump to produce phethylthiomethyl-1-H-isobutryloxychloroformate. A ribonucleoside (10 mmole) was coevaporated 3 times withpyridine and then dried on vacuum pump for 2 hours. Anhydrous pyridine(50 mL) and trimethylsilyl chloride (8.8 ml, 70 mmole) were added, andthe mixture stirred at room temperature for 2 hours.Phethylthiomethyl-1-H-isobutryloxy chloroformate (20 mmole) was thenadded, and stirring continued for another 12 hours. Water (10 mL) wasadded to quench the reaction and hydrolyze trimethylsilyl groups. Thereaction mixture was left overnight. Crude product was evaporated toremove the excess pyridine, and 200 mL of DCM was added with 5% aqueoussolution of NaHCO₃. The precipitated product can be dried and utilizedin the next reactions.

Group V Synthesis of N-(Carbonyloxy-1-methylthiomethylcyclohexane)Ribonucleosides

To a dry 100-mL flask was added 30 mg (0.09 mmol) of anhydrous zinciodine, 30 mg (0.45 mmol) of imidazole, and 5.89 grams of cyclohexanone(60 mmol) of in 15 mL of anhydrous ether. To this stirred solution wasadded 8.1 grams (66 mmol) of methylthiosilane. The reaction was allowedto stir at room temperature for 1 hour and then diluted with 50 mL ofether. The ether was extracted with water and dried over sodium sulfate.The ether was evaporated, and the product distilled at 45° C. at 0.01 mmHg, giving 10.2 grams of1-trimethylsilyloxy-1-methylthiomethylcyclohexane at about 84% yield.

A 250 mL four-necked flask equipped with a stirrer, a thermometer, agas-inlet tube, and a dropping funnel was filled with nitrogen,1-trimethylsilyloxy-1-methylthiomethylcyclo-hexane (10.2 g, 50 mmol),and hexane (100 mL). Through the mixture in the flask, carbon dioxidegas (1.4 liters, 60 mmol) was bubbled at 0° C. using a gas dispersiontube over one hour while stirring. Then, to the resulting slurry typemixture, pyridine (80 mg, 1 mmol) was added at 0° C., and thenmethanesulfonyl chloride (2.87 g, 25 mmol) was dropwise added at thesame temperature, followed by stirring at the same temperature for 2.5hours. After the reaction, 5% sulfuric acid (25 ml) was added to thereaction mixture, and the mixture was stirred for 30 minutes and thenkept standing to separate. The resulting organic layer was washed with a5% aqueous solution of sodium bicarbonate and water successively andconcentrated under reduced pressure at 35 to 40° C. to obtain colorlessliquid bis-(1-methylthiomethylcyclohexane) pyrocarbonate (16.1 g). Theyield was 89%. This product was analyzed by gas chromatography. Thepurity was about 98.6% by GC/MS.

A ribonucleoside (10 mmole) was coevaporated 3 times with pyridine, andthen dried on a vacuum pump for 2 hours. Anhydrous pyridine (50 mL) andtrimethylsilyl chloride (8.8 ml, 70 mmole) were added, and the mixturewas stirred at room temperature for 2 hours.bis-(-1-Methylthiomethylcyclohexane) pyrocarbonate 7.24 grams (20 mmole)was then added, and stirring continued for another 12 hours. Water (10mL) was added to quench the reaction and hydrolyze trimethylsilylgroups. The reaction mixture was left overnight. Crude product wasevaporated to remove the excess pyridine, and 200 mL of DCM was addedwith 5% aqueous solution of NaHCO₃. The precipitated product was driedand utilized in the next reactions.

Group VI Synthesis of N-(4-thiomethylbenzoyl) Ribonucleosides

4-(Methylthio)benzoic acid (50 mmol) was purchased from Aldrich anddissolved in anhydrous hexanes. A large excess of oxalyl chloride(Aldrich) was added to the hexanes solution, and the mixture fitted witha reflux condenser. The reaction was refluxed overnight, and the acidchloride isolated by evaporation.

Ribonucleoside (10 mmole) was coevaporated 3 times with pyridine andthen dried on vacuum pump for 2 hours. Anhydrous pyridine (50 mL) andtrimethylsilyl chloride (6.3 ml, 50 mmole) were added, and the mixturewas stirred at room temperature for 2 hours. 4-thiomethylbenzoylchloride (11 mmole) was then added, and stirring continued for another48 hours. Water (10 mL) was added to quench the reaction and hydrolyzetrimethylsilyl groups. The reaction mixture was left overnight. Crudeproduct was extracted with DCM, washed with 5% aqueous solution ofNaHCO₃, and purified by column chromatography, using CHCI₃ with agradient of methanol (0-5%). The yields were: A 46%; C 56%; and G8%.

Group VII Synthesis of N-(2-thiomethylbenzoyl) Ribonucleosides

2-(Methylthio)benzoic acid (50 mmol) was purchased from Aldrich anddissolved in anhydrous hexanes. A large excess of oxalyl chloride(Aldrich) was added to the hexanes solution, and the mixture fitted witha reflux condenser. The reaction was refluxed overnight, and the2-thiomethylbenzoyl chloride isolated by evaporation.

Ribonucleoside (10 mmole) was coevaporated 3 times with pyridine andthen dried on vacuum pump for 2 hours. Anhydrous pyridine (50 mL) andtrimethylsilyl chloride (6.3 ml, 50 mmole) were added, and the mixturewas stirred at room temperature for 2 hours. 2-Thiomethylbenzoylchloride (11 mmole) was then added, and stirring continued for another48 hours. Water (10 mL) was added to quench the reaction and hydrolyzetrimethylsilyl groups. The reaction mixture was left overnight. Crudeproduct was extracted with DCM, washed with 5% aqueous solution ofNaHCO3, and purified. The yields were: A 44%; C62%; and G31%.

Group VIII Synthesis of N-(2-thiomethylphenoxycarbonyl) Ribonucleosides

2-Thiomethylphenyl chloroformate was made in situ by the reaction of2-(methyl -mercapto)phenol (Aldrich) with a 20% phosgene solution intoluene (Fluka). The phenol was dissolved in anhydrous toluene with anequal molar amount of anhydrous pyridine. The phosgene solution (6 molarequivalents) was cooled on a dry ice/ethanol bath, and the phenolsolution added dropwise. The solution was allowed to warm to roomtemperature and filtered under a blanket of dry argon gas. The resultingclear solution was evaporated to an oil using a rotary evaporatorattached to a Teflon head diaphragm pump. The evaporation processremoved the solvent and excess phosgene. The exhaust from the pump wasbubbled through an aqueous solution of KOH to neutralize the excessphosgene. A ribonucleoside (10 mmole) was coevaporated 3 times withpyridine and then dried on vacuum pump for 2 hours. Anhydrous pyridine(50 mL) and trimethylsilyl chloride (8.8 ml, 70 mmole) were added, andthe mixture was stirred at room temperature for 2 hours.2-Thiomethylphenyl chloroformate (20 mmole) was then added, and stirringcontinued for another 12 hours. Water (10 mL) was added to quench thereaction and hydrolyze trimethylsilyl groups. The reaction mixture wasleft overnight. Crude product was evaporated to remove the excesspyridine, and 200 mL of DCM was added with 5% aqueous solution ofNaHCO₃. The precipitated product was dried and utilized in the nextreactions.

Synthesis of 5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl)N-(2-thiomethylphenoxycarbonyl) Ribonucleosides

5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl) ribonucleoside (10 mmole)was coevaporated 3 times with pyridine, and then dried on vacuum pumpfor 2 hours. Anhydrous pyridine (50 mL) and trimethylsilyl chloride (6.3ml, 50 mmole) were added, and the mixture was stirred at roomtemperature for 2 hours. 2-thiomethylphenyl chloroformate (20 mmole) wasthen added, and stirring continued for another 12 hours. Water (10 mL)was added to quench the reaction and hydrolyze trimethylsilyl groups.The reaction mixture was left overnight. Crude product was extractedwith DCM, washed with 5% aqueous solution of NaHCO₃, and purified bycolumn chromatography using CHCl₃ with a gradient of methanol (0-4%).

Group IX Synthesis of N-(4-thiomethylphenoxycarbonyl) Ribonucleosides

4-Thiomethylphenyl chloroformate was made in situ by the reaction of4-(methyl -mercapto)phenol (Aldrich) with a 20% phosgene solution intoluene (Fluka). The phenol was dissolved in anhydrous toluene with anequal molar amount of anhydrous pyridine. The phosgene solution (6 molarequivalents) was cooled on a dry ice/ethanol bath, and the phenolsolution added dropwise. The solution was allowed to warm to roomtemperature and filtered under a blanket of dry argon gas. The resultingclear solution was evaporated to an oil using a rotary evaporatorattached to a Teflon head diaphragm pump. The evaporation processremoved the solvent and excess phosgene. The exhaust from the pump wasbubbled through an aqueous solution of KOH to neutralize the excessphosgene. A ribonucleoside (10 mmole) was coevaporated 3 times withpyridine and then dried on vacuum pump for 2 hours. Anhydrous pyridine(50 mL) and trimethylsilyl chloride (8.8 ml, 70 mmole) were added, andthe mixture was stirred at room temperature for 2 hours.4-Thiomethylphenyl chloroformate (20 mmole) was then added, and stirringcontinued for another 12 hours. Water (10 mL) was added to quench thereaction and hydrolyze trimethylsilyl groups. The reaction mixture wasleft overnight. Crude product was evaporated to remove the excesspyridine, and 200 mL of DCM was added with 5% aqueous solution ofNaHCO₃. The precipitated product was dried and utilized in the nextreactions.

Synthesis of 5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl)N-(4-thiomethylphenoxy -carbonyl) Ribonucleosides

4-Thiomethylphenyl chloroformate was made in situ by the reaction of4-(methyl -mercapto)phenol (Aldrich) with a 20% phosgene solution intoluene (Fluka). The phenol was dissolved in anhydrous toluene with anequal molar amount of anhydrous pyridine. The phosgene solution (6 molarequivalents) was cooled on a dry ice/ethanol bath, and the phenolsolution added dropwise. The solution was allowed to warn to roomtemperature and filtered under a blanket of dry argon gas. The resultingclear solution was evaporated to an oil using a rotary evaporatorattached to a Teflon head diaphragm pump. The evaporation processremoved the solvent and excess phosgene. The exhaust from the pump wasbubbled through an aqueous solution of KOH to neutralize the excessphosgene. 5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl) ribonucleoside (10mmole) was coevaporated 3 times with pyridine and then dried on vacuumpump for 2 hours. Anhydrous pyridine (50 mL) and trimethylsilyl chloride(6.3 ml, 50 mmole) were added, and the mixture was stirred at roomtemperature for 2 hours. 4-Thiomethylphenyl chloroformate (20 mmole) wasthen added, and stirring continued for another 12 hours. Water (10 mL)was added to quench the reaction and hydrolyze trimethylsilyl groups.The reaction mixture was left overnight. Crude product was extractedwith DCM, washed with 5% aqueous solution of NaHCO₃, and purified bycolumn chromatography using CHCl₃ with a gradient of methanol (0-3%).

Group X Synthesis of N-(t-butylthiocarbamate) Ribonucleosides

t-Butylthiochloroformate was made in situ by the reaction of sodium2-methyl-2-propanethiolate (Aldrich) with a 20% phosgene solution intoluene (Fluka). The sodium 2-methyl-2-propanethiolate was suspended inanhydrous toluene. The phosgene solution (6 molar equivalents) wascooled on a dry ice/ethanol bath, and the sodium2-methyl-2-propanethiolate solution added dropwise. The solution wasallowed to warm to room temperature and filtered under a blanket of dryargon gas. The resulting clear solution was evaporated to an oil using arotary evaporator attached to a Teflon head diaphragm pump. Due to thelow boiling point of the resulting chloroformate, the water bath on therotary evaporator was kept to 20° C. The evaporation process removed thesolvent and excess phosgene. The exhaust from the pump was bubbledthrough an aqueous solution of KOH to neutralize the excess phosgene. Aribonucleoside (10 mmole) was coevaporated 3 times with pyridine andthen dried on vacuum pump for 2 hours. Anhydrous pyridine (50 mL) andtrimethylsilyl chloride (8.8 ml, 70 mmole) were added, and the mixturewas stirred at room temperature for 2 hours. t-Butylthiochloroformate(20 mmole) was then added, and stirring continued for another 12 hours.Water (10 mL) was added to quench the reaction and hydrolyzetrimethylsilyl groups. The reaction mixture was left overnight. Crudeproduct was evaporated to remove the excess pyridine, and 200 mL of DCMwas added with 5% aqueous solution of NaHCO₃. The precipitated productwas dried and utilized in the next reactions. TABLE 1 Deprotection Timeof various APG exocyclic amino Protecting groups in a solution of 5%Hydrogen Peroxide in AMP buffer pH˜9/methanol (50/50, v/v). N4-APG-N6-APG- N2-APG- APG- Cytidine Adenosine Guanosine Phenoxyacetyl <1 min<30 min  <8 hrs 4-t-butylphenoxyacetyl <1 min <30 min  <8 hrs Acetyl <1min <30 min  <8 hrs Chloroacetyl- <1 min <30 min  <8 hrs dichloroacetyl<1 min <30 min  <8 hrs trichloroacetyl <1 min <30 min  <8 hrsFluoroacetyl <1 min <30 min  <8 hrs Difluoroacetyl <1 min <30 min  <8hrs Trifluoroacetyl <1 min <30 min  <8 hrs Nitroacetyl <1 min <30 min <8 hrs n-propionyl <30 min <2 hrs stable n-butyryl <30 min <2 hrsstable i-butyryl <30 min <2 hrs stable n-pentanoyl <30 min <2 hrs stablei-pentanoyl <30 min <2 hrs stable t-pentanoyl <30 min <2 hrs stableMeSCH2CO <60 min <6 hrs >24 hrs PHSCH2CO <60 min <6 hrs >24 hrs2-Cl-PhSCH2CO <60 min <6 hrs >24 hrs 3-Cl-PhSCH2CO <60 min <6 hrs >24hrs 4-Cl-PhSCH2CO <60 min <6 hrs >24 hrs 2-NO2-Benzoyl <60 min <6hrs >24 hrs 3-NO2-Benzoyl <60 min <6 hrs >24 hrs 4-NO2-Benzoyl <60 min<6 hrs >24 hrs 2-Cl-Benzoyl <60 min <6 hrs >24 hrs 3-Cl-Benzoyl <60 min<6 hrs >24 hrs 4-Cl-Benzoyl <60 min <6 hrs >24 hrs 2,4-di-Cl-Benzoyl <60min <6 hrs >24 hrs 2-F-Benzoyl <60 min <6 hrs stable 3-F-Benzoyl <60 min<6 hrs stable 4-F-Benzoyl <60 min <6 hrs stable 2-CF3-Benzoyl <60 min <6hrs >24 hrs 3-CF3-Benzoyl <60 min <6 hrs >24 hrs 4-CF3-Benzoyl <60 min<6 hrs >24 hrs Benzoyl <60 min <6 hrs stable 2-MeO-Benzoyl <2 hrs <24hrs stable 3-MeO-Benzoyl <2 hrs <24 hrs stable 4-MeO-Benzoyl <2 hrs <24hrs stable 2-Me-Benzoyl <2 hrs <24 hrs stable 3-Me-Benzoyl <2 hrs <24hrs stable 4-Me-Benzoyl <2 hrs >24 hrs stable 2,4-di-Me-Benzoyl <6hrs >24 hrs stable 2,4,6-tri-Me-Benzoyl <6 hrs >24 hrs stablet-Butyl-SCO <24 hrs <24 hrs <24 hrs Methyl-SCO <24 hrs <24 hrs <24 hrsEthyl-SCO <24 hrs <24 hrs <24 hrs Propyl-SCO <24 hrs <24 hrs <24 hrsi-propyl-SCO <24 hrs <24 hrs <24 hrs Phenyl-SCO <24 hrs <24 hrs <24 hrs2-Cl-PhSCO <24 hrs <24 hrs <24 hrs 3-Cl-PhSCO <24 hrs <24 hrs <24 hrs4-Cl-PhSCO <24 hrs <24 hrs <24 hrs 2-F-PhSCO <24 hrs <24 hrs <24 hrs3-F-PhSCO <24 hrs <24 hrs <24 hrs 4-F-PhSCO <24 hrs <24 hrs <24 hrs2-CF3-PhSCO <24 hrs <24 hrs <24 hrs 3-CF3-PhSCO <24 hrs <24 hrs <24 hrs4-CF3-PhSCO <24 hrs <24 hrs <24 hrs 2-NO2-PhSCO <24 hrs <24 hrs <24 hrs3-NO2-PhSCO <24 hrs <24 hrs <24 hrs 4-NO2-PhSCO <24 hrs <24 hrs <24 hrs2-OMe-PhSCO <24 hrs <24 hrs <24 hrs 3-OMe-PhSCO <24 hrs <24 hrs <24 hrs4-OMe-PhSCO <24 hrs <24 hrs <24 hrs

2′-Hydroxyl Protecting Group Examples

FIG. 16 illustrates examplary 2′-hydroxyl protective groups.

General procedure for the synthesis of5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-tert-butylthiocarbonate-N-tert-butyl thiocarbonate protected ribonucleosides

5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl) ribonucleoside (20 mmole)was coevaporated 3 times with pyridine and then dried on vacuum pump for12 hours. Anhydrous pyridine (200 mL) and appropriate chloroformate (120mmole) were added, and the mixture was stirred at room temperature for12 hours. The product was purified by column chromatography usinghexanes with a gradient of ethyl acetate (0-60%).

Group I Example 1 Synthesis of5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-(1-oxy-1-methylethyl)1,3-dithiane)protected ribonucleosides

5′, 3′-O-(tetraisopropyldisiloxane-1,3-diyl) ribonucleoside (10 mmole)was coevaporated 3 times with pyridine and then dried on vacuum pump for12 hours. Anhydrous pyridine (100 mL),p-nitrophenyl chloroformate (3.02g, 15 mmole), and DMAP (488 mg, 4 mmole) were added, and the mixture wasstirred at room temperature for 12 hours. The 2′-O-(4-nitrophenylcarbonate) derivate was isolated by flash chromatography, using hexaneswith a gradient of ethyl acetate (0-100%) and then dried on vacuum pumpfor 12 hours.

1,3-Dithiane (4.08 g, 34.00 mmol) in THF (80 mL) was added to n-butyllithium (37.40 mmol) at -78° C. The mixture was allowed to warm to 0° C.on an ice/water bath and then stirred for 30 min. The mixture was onceagain cooled to -78° C., and a solution of freshly distilled acetone(3.74 mL, 50.94 mmol) in anhydrous THF (50 mL) was added drop-wise withstirring. The mixture was allowed to warm to room temperature andstirred to keep the lithium salt of 2-(1-hydroxy-1-methylethyl)1,3-dithiane suspended.

The 5′, 3′-O-(Tetraisopropyldisiloxane-1, 3-diyl) 2′-O-(4-nitrophenylcarbonate) ribonucleoside was redissolved in anhydrous pyridine (75 mL),and the THF solution of 2-(1-hydroxy-1-methylethyl)1, 3-dithiane wasadded. The mixture was stirred at room temperature for 12 hours. Thefinal product was purified by flash chromatography using hexanes with agradient of ethyl acetate (0-30%). The yield was about 74%.

Example 2 Synthesis of 5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(1,1,1,3,3,3-hexafluoro-2-oxy-2-methyl-2-propane)protected ribonucleosides

5′, 3′-O-(Tetraisopropyldisiloxane-1,3-diyl) ribonucleoside (10 mmole)was coevaporated 3 times with pyridine and then dried on vacuum pump for12 hours. Anhydrous pyridine (100 mL),p-nitrophenyl chloroformate (3.02g, 15 mmole), and DMAP (488 mg, 4 mmole) were added, and the mixture wasstirred at room temperature for 12 hours. The 2′-O-(4-nitrophenylcarbonate) derivate was isolated by flash chromatography using hexaneswith a gradient of ethyl acetate (0-100%) and then dried on vacuum pumpfor 12 hours. Anhydrous pyridine (75 mL) and sodium1,1,1,3,3,3-hexafluoro-2-methyl-2-propanolate (1.68 g, 15 mmole) wereadded, and the mixture was stirred at room temperature for 12 hours. Thefinal product was purified by flash chromatography using hexanes with agradient of ethyl acetate (0-30%). The yield was about 22%.

Group Example General produce for the synthesis of5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl) 2′-O-(tert-butylthiocarbonate) protected ribonucleosides (two-step procedure):

5′,3′-O-(Tetraisopropyloxane-1,3-diyl) ribonucleoside (10 mmole) wascoevaporated 3 times with pyridine and then dried on vacuum pump for 12hours. Anhydrous pyridine (100 mL), p-nitrophenyl chloroformate (3.02 g,15 mmole), and DMAP (488 mg, 4 mmole) were added, and the mixture wasstirred at room temperature for 12 hours. The 2′-O-(4-nitrophenylcarbonate) derivate was isolated by flash chromatography using hexaneswith a gradient of ethyl acetate (0-100%) and then dried on vacuum pumpfor 12 hours. Anhydrous pyridine (75 mL) and sodium2-methyl-2-propanethiolate (1.68 g, 15 mmole) were added, and themixture was stirred at room temperature for 12 hours. The final productwas purified by flash chromatography using hexanes with a gradient ofethyl acetate (0-30%). The yields were about 76% for tert-Butylthiocarbonate U analog, about 63% for tert-Butyl thiocarbonate rA-iBuanalog, and 18% for tert-Butyl thiocarbonate rG-AcSMe analog.

General produre for the synthesis of5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl) 2′-O-carbonate/thiocarbonateprotected ribonucleosides (one-step procedure):

5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl) ribonucleoside (20 mmole)was coevaporated 3 times with pyridine and then dried on vacuum pump for12 hours. Anhydrous pyridine (200 mL) and an appropriate chloroformate(120 mmole) were added, and the mixture was stirred at room temperaturefor 12 hours. The product was purified by column chromatography usinghexanes with a gradient of ethyl acetate (0-60%). The yields were about76% for tert-Butyl thiocarbonate U analog and 61% for tert-Butylthiocarbonate rC-Ac analog.

Group III Example 5 Synthesis of 5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-thiomethylacetamide)phenylcarbamateprotected ribonucleosides

2-Nitrophenylaniline (6.9 gm 50 mmol) was dissolved in 100 mL ofanhydrous pyridine. Thiomethyacetyl chloride (55 mmole) was then added,and the reaction stirred for 12 hours. The excess thiomethyacetylchloride was neutralized by the addition of 10 mL of methanol, and thereaction evaporated to dryness. The product, 2-nitro-1-thiomethyl-phenylacetamide, was purified by silica gel flash chromatography inmethylene chloride with a gradient of methanol (0-5%). The product wasconverted to the aniline deravitive using a Raney nickel alloy withammonium chloride in water as described by Bhumik and Akamanchi, (Can.J. Chem. Vol 81, 2003 197-198), which was incorporated herein byreference. The aniline derivative (10 mmol) was converted to theisocyanate in situ by dissolving in toluene (50 mL) with 10% pyridine. A20% solution of phosgene (10 mL) in toluene (Fluka) was placed in a 100mL round bottom flask and cooled to −78° C. The aniline solution wasadded dropwise, and the reaction allowed to warm to 0° C. and stirredovernight. The solution was allowed to warm to room temperature andfiltered under a blanket of dry argon gas. The resulting clear solutionwas evaporated to an oil using a rotary evaporator attached to a Teflonhead diaphragm pump. The excess phosgene and HCl was removed byevaporation. The exhaust from the pump was bubbled through an aqueoussolution of KOH to neutralize the excess phosgene. The resulting crudeisocyanate, 5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl) uridine (3mmole), was coevaporated 3 times with pyridine and then dried on vacuumpump for 2 hours. Anhydrous pyridine (2 mL) and isocyanate (6 mmole)were added, and the mixture was stirred at room temperature for 3 hours.The product was purified by column chromatography using CHCI₃ with agradient of methanol (0-3%). The yields were about 64% for uridine 5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-thiomethylacetamide)phenylcarbamate.

Group IV Example 6 Synthesis of5′-Dimethoxytrityl-2′-O-(2-triisopropylsilyloxy)dimethylphenylmethyl-carbonate protected ribonucleosides

2-Hydroxylacetophenone (15 g, 110 mmol) was dissolved in anhydrousdichloromethane (150 mL) with triethyl amine (250 mmol).Triisopropylsilyl chloride (130 mmol) was dissolved in anhydrousdicholormethane (50 mL) and added to the stirring solution of theacetophenone. The reaction was allowed to stir at room temperatureovernight and was quenched by the addition of water (200 mL). Thedichloromethane layer was separated and dried over sodium sulfate. Thesilylated acetophenone was purified by flash chromatography in hexaneswith an ethyl acetate gradient (0 to 30%). The purified silylatedacetophenone (30 mmol) was redissolved in ether and cooled to 0° C. onan ice/water bath. Methyl magnesium bromide (33 mmol) in ether (1.0 M,Aldrich) was added dropwise to the stirring solution, and the reactionallowed to react for 30 minutes at 0° C. This solution was addeddirectly to a solution of phosgene (30mmol) at −20° C. The phosgenereaction was allowed to stir at −20° C. for 30 min and then added to apyridine solution of 5′-dimethoxytrityl uridine (ChemGenes, Waltham,MA). The reaction was allowed to stir overnight and warm to roomtemperature. The reaction was neutralized by the addition of 10 mL ofwater and evaporated to an oil. The crude reaction was purified directlyon silica gel chromatography using methylene chloride with a methanolgradient (1-4%). Two main products were isolated, including of the 2′and 3′ protected isomers. The yields were about 19% foruridine-5′-DMT-2′-O-(2-triisopropylsilyloxy)dimethylphenylmethylcarbonate.

Group V Example 7 Synthesis of 5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-2-(o-thiomethyl-phenylacetamide)-2-propane carbonate Uridine

2′-Nitroacetophenone was (30 mmol) dissolved in ether and cooled to 0°C. on an ice/water bath. Methyl magnesium bromide (33 mmol) in ether(1.0 M, Aldrich) was added dropwise to the stirring solution, and thereaction allowed to react for 30 minutes at 0° C. A cold aqueoussolution of ammonium chloride was added to the mixture to quench theunreacted methyl magnesium bromide and to protonate the alcoholproducing the 2-(o -nitrophenyl)-2-propanol. The nitrophenyl group wasreduced to the 2-(o-anisyl)-2-propanol by the method described by Bhumikand Akamanchi, (Can. J. Chem. Vol 81, 2003 197-198). The2-(o-anisyl)-2-propanol was purified by silica gel chromatography usingdichloromethane and a methanol gradient. The 2-(o-anisyl)-2-propanol (15mmol) was dissolved in 50 mL of anhydrous pyridine. Thiomethyacetylchloride (15 mmole) was then added, and the reaction stirred for 12hours. The excess thiomethyacetyl chloride was neutralized by theaddition of 5 mL of methanol and the reaction evaporated to dryness. Theproduct, 2-(o-thiomethyl-phenylacetamide)-2-propanol was purified bysilica gel flash chromatography in methylene chloride with a gradient ofmethanol (0-5%). The 2-(o -thiomethylphenylacetamide)-2-propanol (10mmol) was dissolved in THF 30 mL and converted to the sodium salt usingsodium metal. This solution was added directly to a solution of phosgene(10 mmol) at −20° C. The phosgene reaction was allowed to stir at −20°C. for 30 min and then added to a pyridine solution of5′,3′-O-(Tetraisopropyl-disiloxane-1,3-diyl) uridine (Monomer Sciences,New Market, Alabama). The reaction was allowed to stir overnight andwarm to room temperature. The reaction was neutralized by the additionof 10 mL of water and evaporated to an oil. The crude reaction waspurified directly on silica gel chromatography using methylene chloridewith a methanol gradient (1-4%).

Group VI Example 8 Synthesis of5′-Dimethoxytrityl-2′-O-(2-triisopropylsilyloxy)dimethylphenylmethyl-thiocarbonate protected ribonucleosides

2-Hydroxylacetophenone (15 g, 110 mmol) was dissolved in anhydrousdichloromethane (150 mL) with triethyl amine (250 mmol).Triisopropylsilyl chloride (130 mmol) was dissolved in anhydrousdicholormethane (50 mL) and added to the stirring solution of theacetophenone. The reaction was allowed to stir at room temperatureovernight and was quenched by the addition of water (200 mL). Thedichloromethane layer was separated and dried over sodium sulfate. Thesilylated acetophenone was purified by flash chromatography in hexaneswith an ethyl acetate gradient (0 to 30%). The purified silylatedacetophenone (30 mmol) was redissolved in ether and cooled to 0° C. onan ice/water bath. Methyl magnesium bromide (33 mmol) in ether (1.0 M,Aldrich) was added dropwise to the stirring solution, and the reactionallowed to react for 30 minutes at 0° C. A cold aqueous solution ofammonium chloride was added to the mixture to quench the unreactedmethyl magnesium bromide and to protonate the alcohol producing the 2-(o-triisopropylsilyl-oxyphenyl)-2-propanol. The tertiary alcohol was thenconverted to a thiol by the method described by Nishio (J. Chem. Soc.,Chem. Commun., 1989, 4, 205-206), which was incorporated herein byreference. The sodium thiolate was formed using sodium metal in THF andthe resulting solution was added directly to a solution of phosgene (30mmol) at −20° C. The phosgene reaction was allowed to stir at −20° C.for 30 min and then added to a pyridine solution of 5′-dimethoxytrityluridine (ChemGenes, Waltham, Mass). The reaction was allowed to stirovernight and warm to room temperature. The reaction was neutralized bythe addition of 10 mL of water and evaporated to an oil. The crudereaction was purified directly on silica gel chromatography usingmethylene chloride with a methanol gradient (1-4%). Two main productswere isolated consisting of the 2′ and 3′ protected isomers.

Group VII Example 9 Synthesis of 5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-dimethylphenyl-methylthiocarbonate protected ribonucleosides

Sodium 2-phenyl-2-propanethiolate was synthesized from2-phenyl-2-nitropropane by the method described by Komblum and Widmer(J. Am. Chem. Soc., 1987, 100:22, 7086-7088), which was incorporatedherein by reference. 5′,3′-O-(tetraisopropyldisiloxane-1,3-diyl) uridine(10 mmole) was coevaporated 3 times with pyridine and then dried onvacuum pump for 12 hours. Anhydrous pyridine (100 mL),p-nitrophenylchloroformate (3.02 g, 15 mmole) and DMAP (488 mg, 4 mmole) were added,and the mixture was stirred at room temperature for 12 hours.2′-O-(4-nitrophenyl carbonate) derivate was isolated by flashchromatography using hexanes with a gradient of ethyl acetate (0-100%)and then dried on vacuum pump for 12 hours. Anhydrous pyridine (75 mL)and Sodium 2-phenyl-2-propanethiolate (15 mmole) were added, and themixture was stirred at room temperature for 12 hours. The final productwas purified by flash chromatography using hexanes with a gradient ofethyl acetate (0-30%). The yield was about 55% for 5′,3′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-O-dimethylphenyl-methylthiocarbonate uridine.

General Procedure for the Removal of5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl) Protecting Group withHydrogen Fluoride Pyridine Complex

Anhydrous hydrogen fluoride-pyridine (3.5 mL) was carefully added to anice-cold solution of pyridine (4 mL) in MeCN (24 mL). The mixture wasstirred for about 5 minutes and then transferred via cannula to5′,3′-O-(tetraisopropyldisiloxane-1,3-diyl) 2′-O-thiocarbonate protectedribonucleoside (10 mmole). The reaction was left with stirring at roomtemperature for 2-3 hours. Crude reaction mixture (withoutconcentration) was applied to the silica gel, and the product waspurified by column chromatography using hexanes followed by ethylacetate with a gradient of acetone (0-5%).

General Procedure for the Synthesis of 5′-0-(4,4′-dimethoxy-trityl)-2-O-(tert-butyl thiocarbonate)-N-(tert-butylthiocarbonate) Protected Ribonucleosides

The 2′-O-(tert-butyl thiocarbonate)-N-(tert-butyl thiocarbonate)protected ribonucleoside (10 mmole) was coevaporated 3 times withpyridine and then dried on vacuum pump for 12 hours. Anhydrous pyridine(100 mL) and dimethoxytrityl chloride (4.1 g, 12 mmole) were added, andthe mixture was stirred at room temperature for 12 hours. The 5′-O-(4,4′-dimethoxy-trityl)-2′-O-(tert-butyl thiocarbonate)-N-(tert -butylthiocarbonate) derivate was isolated by flash chromatography usinghexanes with a gradient of ethyl acetate (0-100%) and then dried onvacuum pump for 12 hours.

General Procedure for the Synthesis of5′-O-Dimethoxytrityl-2′-O-thiocarbonate Ribonucleoside3′-N,N-diisopropyl(methyl) Phosphoramidites

5′-O-Dimethoxytrityl-2′-O-thiocarbonate ribonucleoside (1 mmole) wasdried on a vacuum pump for 12 hours. Anhydrous THF (3 mL),2,4,6-collidine (0.993 mL, 7.5 mmole) and N-methylimidazole (0.04 mL,0.5 mmole) were added. N,N-diisopropylmethyl phosphonamidic chloride(0.486 mL, 2.5 mmole) was then added dropwise over 10 minutes at RT, andthe reaction mixture was left with stirring for ˜3 hours. Crude mixture(without concentration) was applied on the silica gel, and the productwas purified by column chromatography using benzene with a gradient ofethyl acetate (0-40%).

Synthesis of Oligodeoxyribonucleotides and Oligoribonucleotides.

The solid phase synthesis of oligodeoxyribonucleotides andoligoribonucleotides was accomplished using an ABI model 394 automatedDNA synthesizer from Applied Biosystems (Foster City, Calif.). Thesynthesis cycle was adapted from a standard one-micromolar2-cyanoethyl-phosphoramidite RNA or DNA synthesis cycle. For the ACEchemistry, a separate synthesizer was specially adapted with Teflontubing and fittings to handle the fluoride ion deblock conditions. TheACE chemistry was performed as described by Scaringe et. al. J. Am.Chem. Soc., 1998, 120(45) 11820-11821, which was incorporated herein byreference. The TOM chemistry was performed as described by Pitsch, et.al. in U.S. Pat. No. 5,986,084, which was incorporated herein byreference. RNA was synthesized using the 2′-TBDMS method as described byWincott et. al., Nucleic Acids Research, 1995, 23, 2677-2684, which wasincorporated herein by reference.

Deprotection with Hydrogen Peroxide Solution of Chemically SynthesizedRNA with Commercially available Amino Protecting Groups Example 1

RNA synthesized with 2′-ACE monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with t-butylphenoxyacetyl (Sinha, et. al., Biochimie, 1993,75, 13-23). The solid support was polystyrene containing a peroxideoxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines,and modified the 2′-ACE groups. The 2′-ACE groups were then cleavedusing a buffered aqueous formic acid solution at pH 3.8 overnight.

Example 2

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with t-butylphenoxyacetyl. The solid support was controlledpore glass containing a peroxide oxidizable safety catch linker.Following synthesis, the methyl protecting groups on the phosphodiesterswere cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution and deprotected the exocyclic amines.The RNA containing the 2′-TOM protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 3

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with t-butylphenoxyacetyl. The solid support was thepolystyrene based Rapp Polymere containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This released the RNA oligonucleotides intosolution and deprotected the exocyclic amines. The RNA was then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 4

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with t-butylphenoxyacetyl. The solid support was controlledpore glass containing a peroxide oxidizable safety catch linker.Following synthesis, the methyl protecting groups on the phosphodiesterswere cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, and deprotected the exocyclicamines. The RNA containing the 2′-TBDMS protecting groups were thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture was diluted with water and purified by ion-exchangechromatography.

Example 5

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with t-butylphenoxyacetyl. The solid support was thepolystyrene-based Rapp Polymere containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This released the RNA oligonucleotides intosolution and deprotected the exocyclic amines. The RNA was then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Deprotection of New Amino Protecting Groups (I-X) on ChemicallySynthesized RNA with Hydrogen Peroxide Solution Example 6

RNA was synthesized with 2′-ACE monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-(methylthiomethyloxy-carbonyl). The solid support wasthe polystyrene based Rapp Polymer containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines,and modified the 2′-ACE groups. The 2′-ACE groups were then cleavedusing a buffered aqueous formic acid solution at pH 3.8 overnight.

Example 7

RNA was synthesized with 2′-ACE monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-(methylthiocarbamate). The solid support was thepolystyrene based Rapp Polymer containing a peroxide oxidizable safetycatch linker. The capping step using acetic anhydride was removed fromthe synthesis cycle. Following synthesis, the methyl protecting groupson the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines,and modified the 2′-ACE groups. The 2′-ACE groups were then cleavedusing a buffered aqueous formic acid solution at pH 3.8 overnight.

Example 8

RNA was synthesized with 2′-ACE monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-thiomethylacetyl. The solid support was the polystyrenebased Rapp Polymer containing a peroxide oxidizable safety catch linker.The capping step using acetic anhydride was removed from the synthesiscycle. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines,and modified the 2′-ACE groups. The 2′-ACE groups were then cleavedusing a buffered aqueous formic acid solution at pH 3.8 overnight.

Example 9

RNA was synthesized with 2′-ACE monomers. Cytidine was protected withN-(carbonyloxy-1-phethylthiomethyl-1-H-isobutane), adenosine wasprotected with isobutyryl, and guanosine was protected witht-butylphenoxyacetyl. The solid support was the polystyrene based RappPolymer containing a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasedthe RNA oligonucleotides into solution, deprotected the exocyclicamines, and modified the 2′-ACE groups. The 2′-ACE groups were thencleaved using a buffered aqueous formic acid solution at pH 3.8overnight.

Example 10

RNA was synthesized with 2′-ACE monomers. Cytidine was protected withN-(carbonyloxy-1-methylthiomethylcyclohexane), adenosine was protectedwith isobutyryl, and guanosine was protected with t-butylphenoxyacetyl.The solid support was the polystyrene based Rapp Polymer containing aperoxide oxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines,and modified the 2′-ACE groups. The 2′-ACE groups were then cleavedusing a buffered aqueous formic acid solution at pH 3.8 overnight.

Example 11

RNA was synthesized with 2′-ACE monomers. Cytidine was protected withacetyl, adenosine was protected with N-(4-thiomethylbenzoyl), andguanosine was protected with t-butylphenoxyacetyl. The solid support wasthe polystyrene based Rapp Polymer containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines,and modified the 2′-ACE groups. The 2′-ACE groups were then cleavedusing a buffered aqueous formic acid solution at pH 3.8 overnight.

Example 12

RNA was synthesized with 2′-ACE monomers. Cytidine was protected withacetyl, adenosine was protected with N-(2-thiomethylbenzoyl), andguanosine was protected with t-butylphenoxyacetyl. The solid support wasthe polystyrene based Rapp Polymer containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines,and modified the 2′-ACE groups. The 2′-ACE groups were then cleavedusing a buffered aqueous formic acid solution at pH 3.8 overnight.

Example 13

RNA was synthesized with 2′-ACE monomers. Cytidine was protected withN-(2-thiomethylphenoxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was the polystyrene based Rapp Polymer containing aperoxide oxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines,and modified the 2′-ACE groups. The 2′-ACE groups were then cleavedusing a buffered aqueous formic acid solution at pH 3.8 overnight.

Example 14

RNA was synthesized with 2′-ACE monomers. Cytidine was protected withN-(4-thiomethylphenoxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was the polystyrene based Rapp Polymer containing aperoxide oxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines,and modified the 2′-ACE groups. The 2′-ACE groups were then cleavedusing a buffered aqueous formic acid solution at pH 3.8 overnight.

Example 15

RNA synthesized with 2′-ACE monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-(t-butylthiocarbamate). The solid support was thepolystyrene based Rapp Polymer containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines,and modified the 2′-ACE groups. The 2′-ACE groups were then cleavedusing a buffered aqueous formic acid solution at pH 3.8 overnight.

Example 16

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withN-(methylthiomethyloxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was controlled pore glass containing a peroxide oxidizablesafety catch linker. The acetic anhydride capping step was removed fromthe synthesis cycle. Following synthesis, the methyl protecting groupson the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 17

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withN-(methylthiomethyloxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was the polystyrene based Rapp Polymere containing aperoxide oxidizable safety catch linker. The acetic anhydride cappingstep was removed from the synthesis cycle. Following synthesis, themethyl protecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 18

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-(methylthiocarbamate). The solid support was controlledpore glass containing a peroxide oxidizable safety catch linker. Theacetic anhydride capping step was removed from the synthesis cycle.Following synthesis, the methyl protecting groups on the phosphodiesterswere cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 19

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-(methylthiocarbamate). The solid support was thepolystyrene based Rapp Polymere containing a peroxide oxidizable safetycatch linker. The acetic anhydride capping step was removed from thesynthesis cycle. Following synthesis, the methyl protecting groups onthe phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 20

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-thiomethylacetyl. The solid support was controlled poreglass containing a peroxide oxidizable safety catch linker. The aceticanhydride capping step was removed from the synthesis cycle. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasesthe RNA oligonucleotides into solution and deprotects the exocyclicamines. The RNA containing the 2′-TOM protecting groups was thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture was diluted with water and purified by ion-exchangechromatography.

Example 21

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-thiomethylacetyl . The solid support was thepolystyrene based Rapp Polymere containing. a peroxide oxidizable safetycatch linker. The acetic anhydride capping step was removed from thesynthesis cycle. Following synthesis, the methyl protecting groups onthe phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 22

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withN-(carbonyloxy-1-phethylthiomethyl-1-H-isobutane), adenosine wasprotected with isobutyryl, and guanosine was protected witht-butylphenoxyacetyl. The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasesthe RNA oligonucleotides into solution and deprotects the exocyclicamines. The RNA containing the 2′-TOM protecting groups was thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture was diluted with water and purified by ion-exchangechromatography.

Example 23

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withN-(carbonyloxy-1-phethylthiomethyl-1-H-isobutane), adenosine wasprotected with isobutyryl, and guanosine was protected witht-butylphenoxyacetyl. The solid support was the polystyrene based RappPolymere containing a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a solution of HF/tetraethylene diamine (20% TEMED,10% HF(aq) in acetonitrile at pH 8.6) for 2 hours at room temperature.The fluoride ion solution was washed from the support using acetonitrilefollowed by water. The support was then treated with a 6% hydrogenperoxide solution buffered at pH 9.4 using aminomethylpropanol buffer in10/90 ethanol/water for 4 hours. This releases the RNA oligonucleotidesinto solution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 24

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withN-(carbonyloxy-1-methylthiomethylcyclohexane), adenosine was protectedwith isobutyryl, and guanosine was protected with t-butylphenoxyacetyl.The solid support was controlled pore glass containing a peroxideoxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 25

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withN-(carbonyloxy-1-methylthiomethylcyclohexane), adenosine was protectedwith isobutyryl, and guanosine was protected with t-butylphenoxyacetyl.The solid support was the polystyrene based Rapp Polymere containing aperoxide oxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 26

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, adenosine was protected with N-(4-thiomethylbenzoyl), guanosinewas protected with t-butylphenoxyacetyl. The solid support wascontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 27

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, adenosine was protected with N-(4-thiomethylbenzoyl), guanosinewas protected with t-butylphenoxyacetyl. The solid support was thepolystyrene based Rapp Polymere containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 28

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, adenosine was protected with N-(2-thiomethylbenzoyl), guanosinewas protected with t-butylphenoxyacetyl. The solid support wascontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 29

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, adenosine was protected with N-(2-thiomethylbenzoyl), guanosinewas protected with t-butylphenoxyacetyl. The solid support was thepolystyrene based Rapp Polymere containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 30

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withN-(2-thiomethylphenoxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was controlled pore glass containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 31

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withN-(2-thiomethylphenoxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was the polystyrene based Rapp Polymere containing aperoxide oxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 32

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withN-(4-thiomethylphenoxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was controlled pore glass containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 33

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withN-(4-thiomethylphenoxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was the polystyrene based Rapp Polymere containing aperoxide oxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 34

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-(t-butylthiocarbamate). The solid support wascontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 35

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-(t-butylthiocarbamate). The solid support was thepolystyrene based Rapp Polymere containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 36

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withN-(methylthiomethyloxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was controlled pore glass containing a peroxide oxidizablesafety catch linker. The Acetic Anhydride capping step was removed fromthe synthesis cycle. Following synthesis, the methyl protecting groupson the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 37

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withN-(methylthiomethyloxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was the polystyrene based Rapp Polymere containing aperoxide oxidizable safety catch linker. The acetic anhydride cappingstep was removed from the synthesis cycle. Following synthesis, themethyl protecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 38

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-(methylthiocarbamate). The solid support was controlledpore glass containing a peroxide oxidizable safety catch linker. Theacetic anhydride capping step was removed from the synthesis cycle.Following synthesis, the methyl protecting groups on the phosphodiesterswere cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 39

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-(methylthiocarbamate). The solid support was thepolystyrene based Rapp Polymere containing a peroxide oxidizable safetycatch linker. The acetic anhydride capping step was removed from thesynthesis cycle. Following synthesis, the methyl protecting groups onthe phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 40

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-thiomethylacetyl . The solid support was controlledpore glass containing a peroxide oxidizable safety catch linker. Theacetic anhydride capping step was removed from the synthesis cycle.Following synthesis, the methyl protecting groups on the phosphodiesterswere cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 41

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-thiomethylacetyl. The solid support was the polystyrenebased Rapp Polymere containing a peroxide oxidizable safety catchlinker. The acetic anhydride capping step was removed from the synthesiscycle. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 42

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withN-(carbonyloxy-1-phethylthiomethyl-1-H-isobutane), adenosine wasprotected with isobutyryl, and guanosine was protected witht-butylphenoxyacetyl. The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene- 1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasesthe RNA oligonucleotides into solution and deprotects the exocyclicamines. The RNA containing the 2′-TBDMS protecting groups was thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture was diluted with water and purified by ion-exchangechromatography.

Example 43

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withN-(carbonyloxy-1-phethylthiomethyl-1-H-isobutane), adenosine wasprotected with isobutyryl, and guanosine was protected witht-butylphenoxyacetyl. The solid support was the polystyrene based RappPolymere containing a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a solution of HF/tetraethylene diamine (20% TEMED,10% HF(aq) in acetonitrile at pH 8.6) for 2 hours at room temperature.The fluoride ion solution was washed from the support using acetonitrilefollowed by water. The support was then treated with a 6% hydrogenperoxide solution buffered at pH 9.4 using aminomethylpropanol buffer in10/90 ethanol/water for 4 hours. This releases the RNA oligonucleotidesinto solution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 44

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withN-(carbonyloxy-1-methylthiomethylcyclohexane), adenosine was protectedwith isobutyryl, and guanosine was protected with t-butylphenoxyacetyl.The solid support was controlled pore glass containing a peroxideoxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 45

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withN-(carbonyloxy-1-methylthiomethylcyclohexane), adenosine was protectedwith isobutyryl, and guanosine was protected with t-butylphenoxyacetyl.The solid support was the polystyrene based Rapp Polymere containing aperoxide oxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 46

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, adenosine was protected with N-(4-thiomethylbenzoyl), guanosinewas protected with t-butylphenoxyacetyl. The solid support wascontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF (aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 47

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, adenosine was protected with N-(4-thiomethylbenzoyl), guanosinewas protected with t-butylphenoxyacetyl. The solid support was thepolystyrene based Rapp Polymere containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 48

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, adenosine was protected with N-(2-thiomethylbenzoyl), guanosinewas protected with t-butylphenoxyacetyl. The solid support wascontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 49

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, adenosine was protected with N-(2-thiomethylbenzoyl), guanosinewas protected with t-butylphenoxyacetyl. The solid support was thepolystyrene based Rapp Polymere containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 50

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withN-(2-thiomethylphenoxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was controlled pore glass containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 51

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withN-(2-thiomethylphenoxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was the polystyrene based Rapp Polymere containing aperoxide oxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 52

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withN-(4-thiomethylphenoxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was controlled pore glass containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 53

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withN-(4-thiomethylphenoxycarbonyl), adenosine was protected withisobutyryl, and guanosine was protected with t-butylphenoxyacetyl. Thesolid support was the polystyrene based Rapp Polymere containing aperoxide oxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Example 54

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-(t-butylthiocarbamate). The solid support wascontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups was then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture was dilutedwith water and purified by ion-exchange chromatography.

Example 55

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, adenosine was protected with isobutyryl, and guanosine wasprotected with N-(t-butylthiocarbamate). The solid support was thepolystyrene based Rapp Polymere containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution and deprotects the exocyclic amines. The RNA was directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice, and then isolating by centrifugation.

Deprotection with Hydrogen Peroxide Solution of Chemically SynthesizedRNA on Peroxyanion Cleavable Linker with Commercially AvailableExocyclic Amino Protecting Groups and Novel 2′ Hydroxyl ProtectingGroups Example 56

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. cytidine was protected with acetyl, adenosine was protectedwith isobutyryl, and guanosine was protected withtert-butylphenoxyacetyl. The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 10/90 ethanol/water. This releasedthe RNA oligonucleotides into solution, and deprotected the exocyclicamines and the 2′-BSC groups. The RNA was then directly precipitated byadding 5 volumes of anhydrous ethanol, cooling on dry ice, and thenisolating by centrifugation.

Example 57

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. cytidine was protected with acetyl, adenosine was protectedwith isobutyryl, and guanosine was protected withN-(methylthiomethyloxycarbonyl). The solid support was controlled poreglass containing a peroxide oxidizable safety catch linker. Aceticanhydride capping was removed from the synthesis cycle. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 10/90 ethanol/water. This releasedthe RNA oligonucleotides into solution and deprotected the exocyclicamines and the 2′-BSC groups. The RNA was then directly precipitated byadding 5 volumes of anhydrous ethanol, cooling on dry ice, and thenisolating by centrifugation.

Example 58

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. cytidine was protected with acetyl, adenosine was protectedwith isobutyryl, and guanosine was protected withN-(methylthiocarbamate). The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Acetic anhydridecapping was removed from the synthesis cycle. Following synthesis, themethyl protecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 10/90 ethanol/water. This released the RNAoligonucleotides into solution and deprotected the exocyclic amines andthe 2′-BSC groups. The RNA was then directly precipitated by adding 5volumes of anhydrous ethanol, cooling on dry ice, and then isolating bycentrifugation.

Example 59

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. cytidine was protected with acetyl, adenosine was protectedwith isobutyryl, and guanosine was protected with N-thiomethylacetyl.The solid support was controlled pore glass containing a peroxideoxidizable safety catch linker. Acetic anhydride capping was removedfrom the synthesis cycle. Following synthesis, the methyl protectinggroups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 10/90 ethanol/water. This released the RNAoligonucleotides into solution and deprotected the exocyclic amines andthe 2′-BSC groups. The RNA was then directly precipitated by adding 5volumes of anhydrous ethanol, cooling on dry ice, and then isolating bycentrifugation.

Example 60

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. cytidine was protected with acetyl, adenosine was protectedwith isobutyryl, and guanosine was protected withN-(carbonyloxy-1-phethylthiomethyl-1-H-isobutane). The solid support wascontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 10/90 ethanol/water. This releases the RNAoligonucleotides into solution and deprotects the exocyclic amines andthe 2′-BSC groups. The RNA was then directly precipitated by adding 5volumes of anhydrous ethanol, cooling on dry ice, and then isolating bycentrifugation.

Example 61

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. cytidine was protected with acetyl, adenosine was protectedwith isobutyryl, and guanosine was protected withN-(carbonyloxy-1-methylthiomethylcyclohexane). The solid support wascontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 10/90 ethanol/water. This releases the RNAoligonucleotides into solution and deprotects the exocyclic amines andthe 2′-BSC groups. The RNA was then directly precipitated by adding 5volumes of anhydrous ethanol, cooling on dry ice, and then isolating bycentrifugation.

Example 62

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. cytidine was protected with acetyl, adenosine was protectedwith N-(4-thiomethyl-benzoyl), guanosine was protected withtert-butylphenoxyacetyl. The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 10/90 ethanol/water. This releasedthe RNA oligonucleotides into solution and deprotected the exocyclicamines and the 2′-BSC groups. The RNA was then directly precipitated byadding 5 volumes of anhydrous ethanol, cooling on dry ice, and thenisolating by centrifugation.

Example 63

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. cytidine was protected with acetyl, adenosine was protectedwith N-(2-thiomethyl-benzoyl), guanosine was protected withtert-butylphenoxyacetyl. The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using arninomethylpropanol buffer in 10/90 ethanol/water. This releasedthe RNA oligonucleotides into solution and deprotected the exocyclicamines and the 2′-BSC groups. The RNA was then directly precipitated byadding 5 volumes of anhydrous ethanol, cooling on dry ice, and thenisolating by centrifugation.

Example 64

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. cytidine was protected with N-(2-thiomethylphenoxycarbonyl),adenosine was protected with isobutyryl, and guanosine was protectedwith tert-butylphenoxyacetyl. The solid support was controlled poreglass containing a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 10/90 ethanol/water. This releasedthe RNA oligonucleotides into solution and deprotected the exocyclicamines and the 2′-BSC groups. The RNA was then directly precipitated byadding 5 volumes of anhydrous ethanol, cooling on dry ice, and thenisolating by centrifugation.

Example 65

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. cytidine was protected with N-(4-thiomethylphenoxycarbonyl),adenosine was protected with isobutyryl, and guanosine was protectedwith tert-butylphenoxyacetyl. The solid support was controlled poreglass containing a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 10/90 ethanol/water. This releasedthe RNA oligonucleotides into solution and deprotected the exocyclicamines and the 2′-BSC groups. The RNA was then directly precipitated byadding 5 volumes of anhydrous ethanol, cooling on dry ice, and thenisolating by centrifugation.

Example 66

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. cytidine was protected with acetyl, adenosine was protectedwith isobutyryl, and guanosine was protected withN-(tert-butylthiocarbamate). The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 10/90 ethanol/water. This releasedthe RNA oligonucleotides into solution and deprotected the exocyclicamines and the 2′-BSC groups. The RNA was then directly precipitated byadding 5 volumes of anhydrous ethanol, cooling on dry ice, and thenisolating by centrifugation.

O-4 Protection on Uridine

When a carbonyl protective group was present on the 2′-hydroxyl thatcontains a strong electron withdrawing group, a molecular ion minus 52Dalton (M-52) side product has been observed in the mass spectroscopyanalysis of such RNA products and only associated with the incorporationof uridine. Although not intending to be bound by theory, the productmay be the result of Michael addition at the C-6 carbon of theheterobase followed by nucleophillic acyl substitution at the C-4carbon, resulting in formation of a urea.

FIG. 17 illustrates a Michael addition at the C-6 carbon of theheterobase followed by nucleophillic acyl substitution at the C-4carbon, resulting in formation of a urea. However, this mechanism can beavoided by employing O-4 protection as described below.

FIG. 18 illustrates that an O-4 protection prevents initial Michaeladdition at C-6.

O-4 Protecting on Uridine can be Quite Convenient and high Yielding byEmploying the Formation of a Triazole Deravitive as Described by(Reference)

FIG. 19 illustrates the formation of C-4 triazolide. Because thetriazolide intermediate was quite stable to isolate, it can easily fitinto a regioselective scheme for monomer synthesis.

FIG. 20 illustrates the regiospecific synthesis of 2′-ProtectedNucleoside with O-4 protection.

HPLC Chromatograms of RNA Synthesized by the Present Disclosure

CPG-Q-T-(U^(2′Bsc))₂₀ was synthesized by regular 4-step DMT chemistry onCPG-Q-T using DMTrU^(2′Bsc)OMe phosphoramidite, and then the product wastreated with MeCN/TEMED/HF (4/1/0.5) (40 min), neutralized (TRIS pH7.4), and filtered on Sephadex column. FIG. 21 illustrates fraction 3 onRP HPLC.

CPG-Q-T-rC-rA was synthesized by regular 4-step DMT chemistry withDMTrC^(Ac) _(2′Bsc) OMe phosphoramidite and DMTr^(ibu) _(2′Bsc) OMephosphoramidite. The product was cleaved (off the CPG, the Bsc and Acfrom C, and ibu from A) by 5% H₂O₂ (pH 9.4, 50 mM alkaline buffer, 10%MeOH), and the crude cleavage mixture was analysed by HPLC (regular)(FIG. 22, upper panel) and LC-MS (capillary HPLC) (FIG. 22, on middlepanel). FIG. 22 illustrates the TIC (on the lower panel).

CPG-Q-T-rCrArCrA was synthesized by regular 4-step DMT chemistry withDMTrC^(Ac) _(2′Bsc) OMe phosphoramidite and DMTrA^(ibu) _(2′Bsc) OMephosphoramidite. Analysis was similar to dTrCrA (FIG. 23).

1. A nucleotide monomer comprising: a compound of structure I having a2′-hydroxyl protecting group:

wherein B is selected from a base and a base including a protectinggroup; wherein R₁ and R₂ are each independently selected from H, aprotecting group, and

wherein only one of R₁ and R₂ is

wherein R₃ is selected from an alkyl group, an aryl group, a substitutedalkyl, and a substituted aryl group; wherein R₄ and R₅ are eachindependently selected from an alkyl group, an aryl group, a substitutedalkyl, and a substituted aryl group; and wherein 2HPG is a 2′-hydroxylprotecting group selected from one of the following groups: Group I:

wherein R₆, R₇, and R₈ are each independently selected from an alkylgroup, a substituted alkyl, a substituted alkyl with anelectron-withdrawing group, a substituted aryl group with anelectron-withdrawing group, a group that is converted into an electronwithdrawing group by oxidation, an aryl group, and a substituted arylgroup, wherein R₆, R₇, and R₈ are not each a methyl group, wherein R₆,R₇, and R₈ are not each an unsubstituted alkyl; Group II:

wherein R₆, R₇, and R₈ are each independently selected from an alkylgroup, an aryl group, a substituted alkyl group, and a substituted arylgroup; Group III:

wherein R₉ is selected from an alkyl group, an aryl group, a substitutedalkyl with an electron-withdrawing group, a substituted aryl group withan electron-withdrawing group, wherein R₁₀ and R′₁₀ are eachindependently selected from H, an alkyl group, and a substituted alkylwith an electron-withdrawing group, wherein SG is one or more groups,wherein each SG is bonded to a carbon of the ring, and wherein each SGis selected from H, an alkyl group, an aryl group, a substituted alkylwith an electron-donating group, and a substituted aryl group with anelectron-donating group; Group IVa and IVb:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, asubstituted aryl group with an electron-withdrawing group, wherein R₁₁is selected from an ester protecting group and silyl protecting group,wherein R₁₂ is one or more groups, wherein each R₁₂ is bonded to acarbon on the ring, and wherein each R₁₂ is selected from an alkylgroup, an aryl group, a substituted alkyl with an electron-withdrawinggroup, and a substituted aryl group with an electron-withdrawing group;Group V:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, wherein R₁₃is an amide-protecting group, wherein R₁₂ is one or more groups, whereineach R₁₂ is bonded to a carbon on the ring, and wherein each R₁₂ isselected from an alkyl group, an aryl group, a substituted alkyl with anelectron-withdrawing group, and a substituted aryl group with anelectron-withdrawing group; Group VI:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, whereineach SR₁₄ includes one or more groups, wherein each SR₁₄ is attached toone of the carbons in the carbon ring, wherein SR₁₄ is a thioetherselected from a thioalkyl group, a thioaryl group, a substitutedthioalkyl group, and a substituted thioaryl group, wherein R₁₄ isselected from an alkyl group, an aryl group, a substituted alkyl group,and a substituted aryl group, and wherein each SG includes one or moregroups, wherein each SG is attached to one of the carbons in the carbonring, wherein SG is selected from H, an alkyl group, an aryl group, asubstituted alkyl with an electron-donating group, a substituted arylgroup with an electron-donating group, and an electron-donating group inthe ortho or para position to the thioether SR₁₄; Groups VIIa and VIIb:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, wherein R₁₁is selected from an ester protecting group and a silyl protecting group,wherein each R₁₂ is one or more groups, wherein R₁₂ is bonded to acarbon on the ring, wherein each R₁₂ is selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group; and GroupVIII:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, whereinSR₁₄ includes one or more groups, wherein each SR₁₄ is attached to oneof the carbons in the carbon ring, wherein each SR₁₄ is a thioetherselected from a thioalkyl group, a thioaryl group, a substitutedthioalkyl group, a substituted thioaryl group, wherein R₁₄ is selectedfrom an alkyl group, an aryl group, a substituted alkyl group, asubstituted aryl group, and wherein SG includes one or more groups,wherein each SG is attached to one of the carbons in the carbon ring,wherein each SG is selected from H, an alkyl group, an aryl group, asubstituted alkyl with an electron-donating group, a substituted arylgroup with an electron-donating group, and an electron-donating group inthe ortho or para position to the thioether SR₁₄.
 2. The nucleotidemonomer of claim 1, wherein 2HPG comprises Group I.
 3. The nucleotidemonomer of claim 1, wherein 2HPG comprises Group II.
 4. The nucleotidemonomer of claim 3, wherein R₆, R₇, and R₈ are each a methyl group. 5.The nucleotide monomer of claim 1, wherein 2HPG comprises Group III. 6.The nucleotide monomer of claim 1, wherein 2HPG comprises Group IVa. 7.The nucleotide monomer of claim 1, wherein 2HPG comprises Group IVb. 8.The nucleotide monomer of claim 1, wherein 2HPG comprises Group V. 9.The nucleotide monomer of claim 1, wherein 2HPG comprises Group VI. 10.The nucleotide monomer of claim 1, wherein 2HPG comprises Group VIIa.11. The nucleotide monomer of claim 1, wherein 2HPG comprises GroupVIIb.
 12. The nucleotide monomer of claim 1, wherein 2HPG comprisesGroup VIII.
 13. The nucleotide monomer of claim 1, wherein two or moreof R₆, R₇, and R₈ of Group I are cyclically connected to each other. 14.The nucleotide monomer of claim 1, wherein two or more of R₆, R₇, and R₈of Group II are cyclically connected to each other.
 15. The nucleotidemonomer of claim 1, wherein for Group III R₉ is CF₃, R₁₀ is CH₃, R′₁₀ isH, and SG is H.
 16. The nucleotide monomer of claim 1, wherein forGroups IVa and IVb, R₆ and R₇ are cyclically connected to each other orto the phenyl group
 17. The nucleotide monomer of claim 1, wherein forGroup V, R₆ and R₇ are cyclically connected to each other or to thephenyl group.
 18. The nucleotide monomer of claim 1, wherein for GroupVI, R₆ and R₇ are cyclically connected to each other or to the phenylgroup.
 19. The nucleotide monomer of claim 1, wherein for Groups VIIaand IVIIb, R₆ and R₇ are cyclically connected to each other or to thephenyl group.
 20. The nucleotide monomer of claim 1, wherein for GroupVIII, R₆ and R₇ are cyclically connected to each other or to the phenylgroup.
 21. The nucleotide monomer of claim 1, wherein B is a baseincluding a protection group selected from groups I through X:

wherein Q is selected from one of sulfur (S) and oxygen (0); R isselected from an alkyl group, a substituted alkyl group, an aryl group,and a substituted aryl group; R′ and R″ are independently selected froma halogen, an alkyl group, a substituted alkyl group, an aryl group, anda substituted aryl group; R₁₅ is selected from an alkyl group, an arylgroup, a substituted alkyl, and a substituted aryl group; R₁₆ is one ormore groups, wherein each R₁₆ is attached to one of the carbons in thecarbon ring, wherein each R₁₆ are independently selected from H, ahalogen, a hydroxyl group, an alkoxy group, a substituted alkoxy group,an aryloxy group, a substituted aryloxy group, an amino group, asubstituted amino group, a nitro group, a nitrile group, an alkyl group,an aryl group, a substituted alkyl, and a substituted aryl group,wherein when R₁₆ is more than one, two or more of R₁₆ are optionallycyclically connected to each other; and R₁₇, R₁₈, and R₁₉, are eachindependently selected from H, an alkyl group, an aryl group, asubstituted alkyl, and a substituted aryl group.
 22. A nucleotidemonomer comprising: a structure II having a 2′-hydroxyl protecting groupand a heterobase exocyclic amino protecting group:

wherein R₁ and R₂ are each independently selected from H, a protectinggroup, and

wherein only one of R₁ and R₂ is

wherein R₃ is selected from an alkyl group, an aryl group, a substitutedalkyl, and a substituted aryl group; wherein R₄ and R₅ are eachindependently selected from an alkyl group, an aryl group, a substitutedalkyl, and a substituted aryl group; and wherein 2HPG is identical forboth the 2′-hydroxyl protecting group and the heterobase exocyclic aminoprotected group and selected from one of the following groups: Group I:

wherein R₆, R₇, and R₈ are each independently selected from an alkylgroup, a substituted alkyl, a substituted alkyl with anelectron-withdrawing group, a substituted aryl group with anelectron-withdrawing group, a group that is converted into an electronwithdrawing group by oxidation, an aryl group, and a substituted arylgroup, wherein R₆, R₇, and R₈ are not each a methyl group, wherein R₆,R₇, and R₈ are not each an unsubstituted alkyl; Group II:

wherein R₆, R₇, and R₈ are each independently selected from an alkylgroup, an aryl group, a substituted alkyl group, and a substituted arylgroup; Group III:

wherein R₉ is selected from an alkyl group, an aryl group, a substitutedalkyl with an electron-withdrawing group, a substituted aryl group withan electron-withdrawing group, wherein R₁₀ and R′₁₀ are eachindependently selected from H, an alkyl group, and a substituted alkylwith an electron-withdrawing group, wherein SG is one or more groups,wherein each SG is bonded to a carbon of the ring, and wherein each SGis selected from H, an alkyl group, an aryl group, a substituted alkylwith an electron-donating group, and a substituted aryl group with anelectron-donating group; Group IVa and IVb:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, asubstituted aryl group with an electron-withdrawing group, wherein R₁₁is selected from an ester protecting group and silyl protecting group,wherein R₁₂ is one or more groups, wherein each R₁₂ is bonded to acarbon on the ring, and wherein each R₁₂ is selected from an alkylgroup, an aryl group, a substituted alkyl with an electron-withdrawinggroup, and a substituted aryl group with an electron-withdrawing group;Group V:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, wherein R₁₃is an amide-protecting group, wherein R₁₂ is one or more groups, whereineach R₁₂ is bonded to a carbon on the ring, and wherein each R₁₂ isselected from an alkyl group, an aryl group, a substituted alkyl with anelectron-withdrawing group, and a substituted aryl group with anelectron-withdrawing group; Group VI:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, whereineach SR₁₄ includes one or more groups, wherein each SR₁₄ is attached toone of the carbons in the carbon ring, wherein SR₁₄ is a thioetherselected from a thioalkyl group, a thioaryl group, a substitutedthioalkyl group, and a substituted thioaryl group, wherein R₁₄ isselected from an alkyl group, an aryl group, a substituted alkyl group,and a substituted aryl group, and wherein each SG includes one or moregroups, wherein each SG is attached to one of the carbons in the carbonring, wherein SG is selected from H, an alkyl group, an aryl group, asubstituted alkyl with an electron-donating group, a substituted arylgroup with an electron-donating group, and an electron-donating group inthe ortho or para position to the thioether SR₁₄; Groups VIIa and VIIb:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, wherein R₁₁is selected from an ester protecting group and a silyl protecting group,wherein each R₁₂ is one or more groups, wherein R₁₂ is bonded to acarbon on the ring, wherein each R₁₂ is selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group; and GroupVIII:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, whereinSR₁₄ includes one or more groups, wherein each SR₁₄ is attached to oneof the carbons in the carbon ring, wherein each SR₁₄ is a thioetherselected from a thioalkyl group, a thioaryl group, a substitutedthioalkyl group, a substituted thioaryl group, wherein R₁₄ is selectedfrom an alkyl group, an aryl group, a substituted alkyl group, asubstituted aryl group, and wherein SG includes one or more groups,wherein each SG is attached to one of the carbons in the carbon ring,wherein each SG is selected from H, an alkyl group, an aryl group, asubstituted alkyl with an electron-donating group, a substituted arylgroup with an electron-donating group, and an electron-donating group inthe ortho or para position to the thioether SR₁₄.
 23. The nucleotidemonomer of claim 22, wherein 2HPG comprises Group I
 24. The nucleotidemonomer of claim 22, wherein 2HPG is comprises Group II.
 25. Thenucleotide monomer of claim 24, wherein R6, R7, R8 are each a methylgroup.
 26. The nucleotide monomer of claim 22, wherein 2HPG comprisesGroup III.
 27. The nucleotide monomer of claim 22, wherein 2HPG isselected from a group from one of the following: Group IVa and GroupIVb.
 28. The nucleotide monomer of claim 22, wherein 2HPG comprisesGroup V.
 29. The nucleotide monomer of claim 22, wherein 2HPG comprisesGroup VI.
 30. The nucleotide monomer of claim 22, wherein 2HPG comprisesGroup VIIa.
 31. The nucleotide monomer of claim 22, wherein 2HPGcomprises Group VIIb.
 32. The nucleotide monomer of claim 22, wherein2HPG comprises Group VIII.
 33. The nucleotide monomer of claim 22,wherein two or more of R₆, R₇, and R₈ of group I are cyclicallyconnected to each other.
 34. The nucleotide monomer of claim 22, whereintwo or more of R₆, R₇, and R₈ of group II are cyclically connected toeach other.
 35. The nucleotide monomer of claim 22, wherein for groupIII, R₉ is CF₃, R₁₀ is CH₃, R′₁₀ is H, and SG is H.
 36. The nucleotidemonomer of claim 22, wherein for groups IVa and Ivb, R₆ and R₇ arecyclically connected to each other or to the phenyl group
 37. Thenucleotide monomer of claim 22, wherein for group V, R₆ and R₇ arecyclically connected to each other or to the phenyl group.
 38. Thenucleotide monomer of claim 22, wherein for group VI, R₆ and R₇ arecyclically connected to each other or to the phenyl group.
 39. Thenucleotide monomer of claim 20, wherein for group VIIa, R₆ and R₇ arecyclically connected to each other or to the phenyl group.
 40. Thenucleotide monomer of claim 22, wherein for group VIII, R₆ and R₇ arecyclically connected to each other or to the phenyl group.
 41. Thenucleotide monomer of claim 22, wherein for group IVIIb, R₆ and R₇ arecyclically connected to each other or to the phenyl group.
 42. Thenucleotide monomer of claim 22, wherein B is a base including aprotecting group selected from base protecting groups that are removedunder a same set of conditions as the 2HPG.
 43. The nucleotide monomerof claim 22, wherein B is a base including a protecting group selectedfrom base protecting groups that are removed under a different set ofconditions than the 2HPG.
 44. The nucleotide monomer of claim 22,wherein B is a base including a protecting group selected from: acetyl,chloroacetyl, dicholoracetyl, trichloroacetyl, fluoroacetyl,difluoroacetyl, trifluoroacetyl, nitroacetyl, propionyl, n-butyryl,i-butyryl, n-pentanoyl, i-pentanoyl, t-pentanoyl, phenoxyacetyl,2-chlorophenoxyacetyl, t-butyl-phenoxyacetyl, methylthioacetyl,phenylthioacetyl, 2-chlorophenylthioacetyl, 3-chlorophenylthioacetyl,4-chlorophenylthioacetyl, t-butyl-phenylthioacetyl, benzyloxycarbonyl,(9-fluoroenyl)-methoxycarbonyl (Fmoc), 2-nitrophenylsulfenyl,4-nitrophenylethylcarbonyl, 4-nitrophenylethoxycarbonyl,diphenylcarbamoyl, morpholinocarbamoyl, dialkylformamidines, succinyl,phtaloyl, benzoyl, 4-trifluoromethylbenzoyl, 2-methylbenzoyl,3-methylbenzoyl, 4-methylbenzoyl, 2, 4-dimethylbenzoyl, 2,6-dimethylbenzoyl, 2, 4, 6-trimethylbenzoyl, 2-methoxybenzoyl,3-methoxybenzoyl, 4-methoxybenzoyl, 2, 4-dimethoxybenzoyl, 2,6-dimethoxybenzoyl, 2, 4, 6-trimethoxybenzoyl, 2-methylthiobenzoyl, 3-methylthiobenzoyl, 4- methylthiobenzoyl, 2, 4-dimethylthiobenzoyl, 2,6-dimethylthiobenzoyl, 2, 4, 6-trimethylthiobenzoyl, 2-chlorobenzoyl,3-chlorobenzoyl, 4-chlorobenzoyl, 2, 4-dichlorobenzoyl, 2,6-dicholorbenzoyl, 2-fluorobenzoyl, 3-fluorobenzoyl, 4-fluorobenzoyl, 2,4-difluorobenzoyl, 2, 6-difluorobenzoyl, 2, 5-difluorobenzoyl, 3,5-difluorobenzoyl, 2-trifluoromethylbenzoyl, 3-trifluoromethylbenzoyl,2, 4- trifluoromethylbenzoyl, 2, 6-trifluoromethylbenzoyl, 2,5-trifluoromethylbenzoyl, 3, 5-trifluoromethylbenzoyl, 2-nitrobenzoyl,3-nitrobenzoyl, 4-nitrobenzoyl, (3-methoxy 4-phenoxy)benzoyl,(triphenyl)silylethyleneoxycarbonyl,(diphenylmethyl)silylethyleneoxycarbonyl,(phenyldimethyl)silylethyleneoxycarbonyl,(trimethy)lsilylethyleneoxycarbonyl,(triphenyl)silyl(2,2-dimethyl)ethyleneoxycarbonyl,(diphenylmethyl)silyl[(2,2-dimethyl)ethylene]oxycarbonyl,phenyldimethylsilyl[(2,2-dimethyl)ethylene]oxycarbonyl,trimethylsilyl[(2,2-dimethyl)ethylene]oxycarbonyl, methyloxycarbonyl,ethyloxycarbonyl, propyloxycarbonyl, isopropyloxycarbonyl,butyloxycarbonyl, isobutyloxycarbonyl, t-butyloxycarbonyl,phenyloxycarbonyl, benzyloxycarbonyl, methylthiocarbonyl,ethylthiocarbonyl, propylthiocarbonyl, isopropylthiocarbonyl,butylthiocarbonyl, isobutylthiocarbonyl, tert-butylthiocarbonyl,phenylthiocarbonyl, benzylthiocarbonyl, methyloxymethyleneoxycarbonyl,methylthiomethylene-oxycarbonyl, phenyloxymethylene-oxycarbonyl,phenyllthiomethyleneoxycarbonyl, methyloxy(methyl)methyleneoxycarbonyl,methylthio(methyl)methyleneoxycarbonyl,methyloxy(dimethyl)methyleneoxycarbonyl,methylthio(dimethyl)methyleneoxycarbonyl,phenyloxy(methyl)methyleneoxycarbonyl,phenylthio(methyl)methyleneoxycarbonyl,phenyloxy(dimethyl)methyleneoxycarbonyl,phenylthio(dimethyl)methyleneoxycarbonyl, and substituted derivatives ofthese groups.
 45. A polynucleotide compound comprising:

wherein B is selected from a base including a protection group; whereinR₃ is selected from an alkyl group, an aryl group, a substituted alkyl,and a substituted aryl group; wherein 2HPG is a 2′-hydroxyl protectinggroup selected from one of the following groups: Group I:

wherein R₆, R₇, and R₈ are each independently selected from an alkylgroup, a substituted alkyl, a substituted alkyl with anelectron-withdrawing group, a substituted aryl group with anelectron-withdrawing group, a group that is converted into an electronwithdrawing group by oxidation, an aryl group, and a substituted arylgroup, wherein R₆, R₇, and R₈ are not each a methyl group, wherein R₆,R₇, and R₈ are not each an unsubstituted alkyl; Group II:

wherein R₆, R₇, and R₈ are each independently selected from an alkylgroup, an aryl group, a substituted alkyl group, and a substituted arylgroup; Group III:

wherein R₉ is selected from an alkyl group, an aryl group, a substitutedalkyl with an electron-withdrawing group, a substituted aryl group withan electron-withdrawing group, wherein R₁₀ and R′₁₀ are eachindependently selected from H, an alkyl group, and a substituted alkylwith an electron-withdrawing group, wherein SG is one or more groups,wherein each SG is bonded to a carbon of the ring, and wherein each SGis selected from H, an alkyl group, an aryl group, a substituted alkylwith an electron-donating group, and a substituted aryl group with anelectron-donating group; Group IVa and IVb:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, asubstituted aryl group with an electron-withdrawing group, wherein R₁₁is selected from an ester protecting group and silyl protecting group,wherein R₁₂ is one or more groups, wherein each R₁₂ is bonded to acarbon on the ring, and wherein each R₁₂ is selected from an alkylgroup, an aryl group, a substituted alkyl with an electron-withdrawinggroup, and a substituted aryl group with an electron-withdrawing group;Group V:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, wherein R₁₃is an amide-protecting group, wherein R₁₂ is one or more groups, whereineach R₁₂ is bonded to a carbon on the ring, and wherein each R₁₂ isselected from an alkyl group, an aryl group, a substituted alkyl with anelectron-withdrawing group, and a substituted aryl group with anelectron-withdrawing group; Group VI:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, whereineach SR₁₄ includes one or more groups, wherein each SR₁₄ is attached toone of the carbons in the carbon ring, wherein SR₁₄ is a thioetherselected from a thioalkyl group, a thioaryl group, a substitutedthioalkyl group, and a substituted thioaryl group, wherein R₁₄ isselected from an alkyl group, an aryl group, a substituted alkyl group,and a substituted aryl group, and wherein each SG includes one or moregroups, wherein each SG is attached to one of the carbons in the carbonring, wherein SG is selected from H, an alkyl group, an aryl group, asubstituted alkyl with an electron-donating group, a substituted arylgroup with an electron-donating group, and an electron-donating group inthe ortho or para position to the thioether SR₁₄; Groups VIIa and VIIb:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, wherein R₁₁is selected from an ester protecting group and a silyl protecting group,wherein each R₁₂ is one or more groups, wherein R₁₂ is bonded to acarbon on the ring, wherein each R₁₂ is selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group; and GroupVIII:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, whereinSR₁₄ includes one or more groups, wherein each SR₁₄ is attached to oneof the carbons in the carbon ring, wherein each SR₁₄ is a thioetherselected from a thioalkyl group, a thioaryl group, a substitutedthioalkyl group, a substituted thioaryl group, wherein R₁₄ is selectedfrom an alkyl group, an aryl group, a substituted alkyl group, asubstituted aryl group, and wherein SG includes one or more groups,wherein each SG is attached to one of the carbons in the carbon ring,wherein each SG is selected from H, an alkyl group, an aryl group, asubstituted alkyl with an electron-donating group, a substituted arylgroup with an electron-donating group, and an electron-donating group inthe ortho or para position to the thioether SR₁₄.
 46. The polynucleotideof claim 46, wherein 2HPG comprises Group I.
 47. The polynucleotide ofclaim 46, wherein 2HPG comprises Group II.
 48. The polynucleotide ofclaim 47, wherein R₆, R₇, and R₈ are each a methyl group.
 49. Thepolynucleotide of claim 45, wherein 2HPG comprises Group III.
 50. Thepolynucleotide of claim 45, wherein 2HPG is selected from a group in oneof the following: Group IVa and Group IVb.
 51. The polynucleotide ofclaim 45, wherein 2HPG comprises Group V.
 52. The polynucleotide ofclaim 45, wherein 2HPG comprises Group VI.
 53. The polynucleotide ofclaim 45, wherein 2HPG comprises Group VIIa.
 54. The polynucleotide ofclaim 45, wherein 2HPG comprises Group VIII.
 55. The polynucleotide ofclaim 45, wherein 2HPG comprises Group VIIb.
 56. The polynucleotide ofclaim 45, wherein two or more of R₆, R₇, and R₈ of group I arecyclically connected to each other.
 57. The polynucleotide of claim 45,wherein two or more of R₆, R₇, and R₈ of group II are cyclicallyconnected to each other.
 58. The polynucleotide of claim 45, wherein forgroup III, R₉is CF₃, R₁₀ is CH₃, R′₁₀ is H, and SG is H.
 59. Thepolynucleotide of claim 45, wherein for groups IVa and Ivb, R₆ and R₇are cyclically connected to each other or to the phenyl group
 60. Thepolynucleotide of claim 45, wherein for group V, R₆ and R₇ arecyclically connected to each other or to the phenyl group.
 61. Thepolynucleotide of claim 45, wherein for group VI, R₆ and R₇ arecyclically connected to each other or to the phenyl group.
 62. Thepolynucleotide of claim 45, wherein for groups VIIa and IVIIb, R₆ and R₇are cyclically connected to each other or to the phenyl group.
 63. Thepolynucleotide of claim 45, wherein for group VIII, R₆ and R₇ arecyclically connected to each other or to the phenyl group.
 64. Thepolynucleotide of claim 45, wherein the polynucleotide is a ribonucleicacid.
 65. The polynucleotide compound of claim 46, wherein B is a baseincluding a protection group selected from groups I through X:

wherein Q is selected from one of sulfur (S) and oxygen (O); R isselected from an alkyl group, a substituted alkyl group, an aryl group,and a substituted aryl group; R′ and R″ are independently selected froma halogen, an alkyl group, a substituted alkyl group, an aryl group, anda substituted aryl group; R₁₅ is selected from an alkyl group, an arylgroup, a substituted alkyl, and a substituted aryl group; R₁₆ is one ormore groups, wherein each R₁₆ is attached to one of the carbons in thecarbon ring, wherein each R₁₆ is independently selected from H, ahalogen, a hydroxyl group, an alkoxy group, a substituted alkoxy group,an aryloxy group, a substituted aryloxy group, an amino group, asubstituted amino group, a nitro group, a nitrile group, an alkyl group,an aryl group, a substituted alkyl, and a substituted aryl group,wherein when R₁₆ is more than one, two or more of R₁₆ are optionallycyclically connected to each other; and R₁₇, R₁₈, and R₁₉, are eachindependently selected from H, an alkyl group, an aryl group, asubstituted alkyl, and a substituted aryl group.
 66. The polynucleotidecompound of claim 45, wherein B is a base including a protection groupselected from base protecting groups that are removed under a same setof conditions than the 2HPG.
 67. The polynucleotide compound of claim45, wherein B is a base including a protection group selected from baseprotecting groups that are removed under a different set of conditionsas the 2HPG.
 68. The polynucleotide compound of claim 45, wherein B is abase including a protection group selected from: acetyl, chloroacetyl,dicholoracetyl, trichloroacetyl, fluoroacetyl, difluoroacetyl,trifluoroacetyl, nitroacetyl, propionyl, n-butyryl, i-butyryl,n-pentanoyl, i-pentanoyl, t-pentanoyl, phenoxyacetyl,2-chlorophenoxyacetyl, t-butyl-phenoxyacetyl, methylthioacetyl,phenylthioacetyl, 2-chlorophenylthioacetyl, 3-chlorophenylthioacetyl,4-chlorophenylthioacetyl, t-butyl-phenylthioacetyl, benzyloxycarbonyl,(9-fluoroenyl)-methoxycarbonyl (Fmoc), 2-nitrophenylsulfenyl,4-nitrophenylethylcarbonyl, 4-nitrophenylethoxycarbonyl,diphenylcarbamoyl, morpholinocarbamoyl, dialkylformamidines, succinyl,phtaloyl, benzoyl, 4-trifluoromethylbenzoyl, 2-methylbenzoyl,3-methylbenzoyl, 4-methylbenzoyl, 2, 4-dimethylbenzoyl, 2,6-dimethylbenzoyl, 2, 4, 6-trimethylbenzoyl, 2-methoxybenzoyl,3-methoxybenzoyl, 4-methoxybenzoyl, 2, 4-dimethoxybenzoyl, 2,6-dimethoxybenzoyl, 2, 4, 6-trimethoxybenzoyl, 2-methylthiobenzoyl, 3-methylthiobenzoyl, 4- methylthiobenzoyl, 2, 4-dimethylthiobenzoyl, 2,6-dimethylthiobenzoyl, 2, 4, 6-trimethylthiobenzoyl, 2-chlorobenzoyl,3-chlorobenzoyl, 4-chlorobenzoyl, 2, 4-dichlorobenzoyl, 2,6-dicholorbenzoyl, 2-fluorobenzoyl, 3-fluorobenzoyl, 4-fluorobenzoyl, 2,4-difluorobenzoyl, 2, 6-difluorobenzoyl, 2, 5-difluorobenzoyl, 3,5-difluorobenzoyl, 2-trifluoromethylbenzoyl, 3-trifluoromethylbenzoyl,2, 4- trifluoromethylbenzoyl, 2, 6- trifluoromethylbenzoyl, 2, 5-trifluoromethylbenzoyl, 3, 5-trifluoromethylbenzoyl, 2-nitrobenzoyl,3-nitrobenzoyl, 4-nitrobenzoyl, (3-methoxy 4-phenoxy)benzoyl,(triphenyl)silylethyleneoxycarbonyl,(diphenylmethyl)silylethyleneoxycarbonyl,(phenyldimethyl)silylethyleneoxycarbonyl,(trimethy)lsilylethyleneoxycarbonyl,(triphenyl)silyl(2,2-dimethyl)ethyleneoxycarbonyl,(diphenylmethyl)silyl[(2,2-dimethyl)ethylene]oxycarbonyl,phenyldimethylsilyl[(2,2-dimethyl)ethylene]oxycarbonyl,trimethylsilyl[(2,2-dimethyl)ethylene]oxycarbonyl, methyloxycarbonyl,ethyloxycarbonyl, propyloxycarbonyl, isopropyloxycarbonyl,butyloxycarbonyl, isobutyloxycarbonyl, t-butyloxycarbonyl,phenyloxycarbonyl, benzyloxycarbonyl, methylthiocarbonyl,ethylthiocarbonyl, propylthiocarbonyl, isopropylthiocarbonyl,butylthiocarbonyl, isobutylthiocarbonyl, tert-butylthiocarbonyl,phenylthiocarbonyl, benzylthiocarbonyl, methyloxymethyleneoxycarbonyl,methylthiomethylene-oxycarbonyl, phenyloxymethylene-oxycarbonyl,phenyllthiomethyleneoxycarbonyl, methyloxy(methyl)methyleneoxycarbonyl,methylthio(methyl)methyleneoxycarbonyl,methyloxy(dimethyl)methyleneoxycarbonyl,methylthio(dimethyl)methyleneoxycarbonyl,phenyloxy(methyl)methyleneoxycarbonyl,phenylthio(methyl)methyleneoxycarbonyl,phenyloxy(dimethyl)methyleneoxycarbonyl,phenylthio(dimethyl)methyleneoxycarbonyl, and substituted derivatives ofthese groups.
 69. A method of deprotecting polynucleotides comprising:synthesizing on a solid support a polynucleotide of claim 45;introducing the polynucleotide to a solution including an α-effectnucleophile, wherein the solution is at a pH of about 6 to 11, whereinthe α-effect nucleophile has a pKa of about 4 to 13; and deprotectingthe 2′-hydroxyl protecting group.
 70. The method of claim 69, furthercomprising precipitating the polynucleotide out of the solution.
 71. Themethod of claim 69, wherein B is a base including a protecting group.72. The method of claim 69, wherein the exocyclic amino protecting groupand the 2′-hydroxyl protecting group are deprotected simultaneously. 73.The method of claim 69, wherein the exocyclic amino protecting group andthe 2′-hydroxyl protecting group are deprotected independently.
 74. Themethod of claim 69, further comprising cleaving the polynucleotide fromthe support.
 75. The method of claim 69, wherein the support is anarray.
 76. A polynucleotide compound comprising:

wherein B is selected from a base and a base including protecting group,wherein each protecting group is selected from one of the following: anexocyclic amino protection group and an imino protecting group; whereinR₃ is selected from an alkyl group, an aryl group, a substituted alkyl,and a substituted aryl group; wherein 2HPG is identical for both the2′-hydroxyl protecting group and the heterobase exocyclic aminoprotecting group sand is selected from one of the following groups:Group I:

wherein R₆, R₇, and R₈ are each independently selected from an alkylgroup, a substituted alkyl, a substituted alkyl with anelectron-withdrawing group, a substituted aryl group with anelectron-withdrawing group, a group that is converted into an electronwithdrawing group by oxidation, an aryl group, and a substituted arylgroup, wherein R₆, R₇, and R₈ are not each a methyl group, wherein R₆,R₇, and R₈ are not each an unsubstituted alkyl; Group II:

wherein R₆, R₇, and R₈ are each independently selected from an alkylgroup, an aryl group, a substituted alkyl group, and a substituted arylgroup; Group III:

wherein R₉ is selected from an alkyl group, an aryl group, a substitutedalkyl with an electron-withdrawing group, a substituted aryl group withan electron-withdrawing group, wherein R₁₀ and R′₁₀ are eachindependently selected from H, an alkyl group, and a substituted alkylwith an electron-withdrawing group, wherein SG is one or more groups,wherein each SG is bonded to a carbon of the ring, and wherein each SGis selected from H, an alkyl group, an aryl group, a substituted alkylwith an electron-donating group, and a substituted aryl group with anelectron-donating group; Group IVa and IVb:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, asubstituted aryl group with an electron-withdrawing group, wherein R₁₁is selected from an ester protecting group and silyl protecting group,wherein R₁₂ is one or more groups, wherein each R₁₂ is bonded to acarbon on the ring, and wherein each R₁₂ is selected from an alkylgroup, an aryl group, a substituted alkyl with an electron-withdrawinggroup, and a substituted aryl group with an electron-withdrawing group;Group V:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, wherein R₁₃is an amide-protecting group, wherein R₁₂ is one or more groups, whereineach R₁₂ is bonded to a carbon on the ring, and wherein each R₁₂ isselected from an alkyl group, an aryl group, a substituted alkyl with anelectron-withdrawing group, and a substituted aryl group with anelectron-withdrawing group; Group VI:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, whereineach SR₁₄ includes one or more groups, wherein each SR₁₄ is attached toone of the carbons in the carbon ring, wherein SR₁₄ is a thioetherselected from a thioalkyl group, a thioaryl group, a substitutedthioalkyl group, and a substituted thioaryl group, wherein R₁₄ isselected from an alkyl group, an aryl group, a substituted alkyl group,and a substituted aryl group, and wherein each SG includes one or moregroups, wherein each SG is attached to one of the carbons in the carbonring, wherein SG is selected from H, an alkyl group, an aryl group, asubstituted alkyl with an electron-donating group, a substituted arylgroup with an electron-donating group, and an electron-donating group inthe ortho or para position to the thioether SR₁₄; Groups VIIa and VIIb:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, wherein R₁₁is selected from an ester protecting group and a silyl protecting group,wherein each R₁₂ is one or more groups, wherein R₁₂ is bonded to acarbon on the ring, wherein each R₁₂ is selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group; and GroupVIII:

wherein R₆ and R₇ are independently selected from an alkyl group, anaryl group, a substituted alkyl with an electron-withdrawing group, anda substituted aryl group with an electron-withdrawing group, whereinSR₁₄ includes one or more groups, wherein each SR₁₄ is attached to oneof the carbons in the carbon ring, wherein each SR₁₄ is a thioetherselected from a thioalkyl group, a thioaryl group, a substitutedthioalkyl group, a substituted thioaryl group, wherein R₁₄ is selectedfrom an alkyl group, an aryl group, a substituted alkyl group, asubstituted aryl group, and wherein SG includes one or more groups,wherein each SG is attached to one of the carbons in the carbon ring,wherein each SG is selected from H, an alkyl group, an aryl group, asubstituted alkyl with an electron-donating group, a substituted arylgroup with an electron-donating group, and an electron-donating group inthe ortho or para position to the thioether SR₁₄.
 77. The polynucleotideof claim 76, wherein 2HPG comprises Group I.
 78. The polynucleotide ofclaim 76, wherein 2HPG comprises Group II.
 79. The polynucleotide ofclaim 78, wherein R6, R7, and R8 are each a methyl group.
 80. Thepolynucleotide of claim 76, wherein 2HPG comprises Group III.
 81. Thepolynucleotide of claim 76, wherein 2HPG is selected from a group in oneof the following: Group IVa and Group IVb.
 82. The polynucleotide ofclaim 76, wherein 2HPG comprises Group V.
 83. The polynucleotide ofclaim 76, wherein 2HPG comprises Group VI.
 84. The polynucleotide ofclaim 76, wherein 2HPG is selected from a group in one of the following:Group VIIa and Group VIIb.
 85. The polynucleotide of claim 76, wherein2HPG comprises Group VIII.
 86. The polynucleotide of claim 76, whereinthe polynucleotide is a ribonucleic acid.
 87. The polynucleotide ofclaim 76, wherein two or more of R₆, R₇, and R₈ of group I arecyclically connected to each other.
 88. The polynucleotide of claim 76,wherein two or more of R₆, R₇, and R₈ of group II are cyclicallyconnected to each other.
 89. The polynucleotide of claim 76, wherein forgroup III, R₉ is CF₃, R₁₀ is CH₃, R′₁₀ is H, and SG is H.
 90. Thepolynucleotide of claim 76, wherein for groups IVa and IVb, R₆ and R₇are cyclically connected to each other or to the phenyl group
 91. Thepolynucleotide of claim 76, wherein for group V, R₆ and R₇ arecyclically connected to each other or to the phenyl group.
 92. Thepolynucleotide of claim 76, wherein for group VI, R₆ and R₇ arecyclically connected to each other or to the phenyl group.
 93. Thepolynucleotide of claim 76, wherein for groups VIIa and IVIIb, R₆ and R₇are cyclically connected to each other or to the phenyl group.
 94. Thepolynucleotide of claim 76, wherein for group VIII, R₆ and R₇ arecyclically connected to each other or to the phenyl group.
 95. A methodof deprotecting polynucleotides, comprising: providing a syntheticallymade polynucleotide of claim 76; and deprotecting the 2′-hydroxylprotecting group and the exocyclic amino protecting group in a solutionincluding an α-effect nucleophile, wherein the solution is at a pH ofabout 4 to 10, and wherein the α-effect nucleophile has a pKa of about 4to
 13. 96. The method of claim 95, wherein the α-effect nucleophile is aperoxyanion.
 97. The method of claim 95, wherein the α-effectnucleophile is selected from hydrogen peroxide and salts thereof,peracids and salts thereof, perboric acids and salts thereof,alkylperoxides and salts thereof, hydroperoxides and salts thereof,butylhydroperoxide and salts thereof, benzylhydroperoxide and saltsthereof, phenylhydroperoxide and salts thereof, performic acid and saltsthereof, peracetic acid and salts thereof, perbenzoic acid and saltsthereof, chloroperbenzoic acid and salts thereof, and combinationsthereof.
 98. A process for the preparation of a ribonucleotide monomerof claim 22, comprising: reacting a ribonucleoside of formula:

wherein PG1 and PG2 are hydroxyl protecting groups; with a pyrocarbonatederivative of the formula: Rp—O—Rp, wherein Rp are individually selectedfrom compounds that include a group selected from: Group I, Group IVa,Group IVb, Group V, and Group VI; deblocking PG1 and PG2; protecting the5′-hydroxyl moiety with a hydroxyl protecting group; and phosphitylatingthe 3′-hydroxyl moiety to produce the monomers of claim
 22. 99. Theprocess of claim 98, wherein PG1 and PG2 are eachtetraisopropyldisiloxane-1,3-diyl (TIPS), and wherein after deprotectionof TIPS with fluoride anions, the 5′-hydroxyl is further protected withDMT, and the 3′-hydroxyl is further protected with 3′-O-(methyl)-N,N-diisopropylphopshoramidite.
 100. A process for thepreparation of a ribonucleotide monomer of claim 22, comprising:reacting a ribonucleoside of formula:

wherein PG1 and PG2 are hydroxyl protecting groups; with athiochloroformate derivative of the formula: Cl-S-Rt, wherein Rt is acompound selected from: Group II, Group V, Group VIIa, Group VIIB, GroupVIII and Group III, and wherein R10 and R′10 are not hydrogen;deblocking PG1 and PG2; protecting the 5′-hydroxyl moiety with ahydroxyl protecting group; and phosphitylating the 3′-hydroxyl moiety toproduce the monomers of claim
 22. 101. A process for the preparation ofa ribonucleotide monomer of claim 22, comprising: reacting aribonucleoside of formula:

wherein PG1 and PG2 are hydroxyl protecting groups; with an isocyanatederivative of formula

wherein R₉ is a group selected from: an alkyl group, an aryl group, asubstituted alkyl with an electron-withdrawing group, a substituted arylgroup with an electron-withdrawing group, wherein R₁₀ and R′₁₀ are eachindependently selected from: H, an alkyl group, and a substituted alkylwith an electron-withdrawing group, wherein SG is one or more groups,wherein each SG is bonded to a carbon of the carbon ring, and whereineach SG is selected from one or more substituent groups independentlyselected from H, an alkyl group, an aryl group, a substituted alkyl withan electron-donating group, and a substituted aryl group with anelectron-donating group; deblocking PG1 and PG2; protecting the5′-hydroxyl moiety with a hydroxyl protecting group; and phosphitylatingthe 3′-hydroxyl moiety to produce the monomers of claim
 22. 102. Aprocess for the preparation of a ribonucleotide monomer of claim 22,comprising: reacting a ribonucleoside of formula:

wherein PG1 and PG2 are hydroxyl protecting groups; with Cl-Ri, whereinRi is selected from Group III; deblocking PG1 and PG2; protecting the5′-hydroxyl moiety with a hydroxyl protecting group; and phosphitylatingthe 3′-hydroxyl moiety to produce the monomers of claim
 22. 103. Amethod for the chemical synthesis of a polynucleotide with apredetermined nucleotide-sequence comprising: coupling a nucleotidemonomer of structure I of claim 1, wherein R₂ is a phosphoramidite, witha terminally deprotected polynucleotide precursor.
 104. A method for thechemical synthesis of a polynucleotide with a predeterminednucleotide-sequence comprising: coupling a nucleotide monomer ofstructure II of claim 22, wherein R₂ is a phosphoramidite, with aterminally deprotected polynucleotide precursor.